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Class 
Book. 







CopightN , 



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COPYRIGHT DEPOSIT. 



PRACTICAL CLINICAL 

LABORATORY DIAGNOSIS 

A THOROUGHLY ILLUSTRATED LABORATORY GUIDE 

EMBODYING THE INTERPRETATION OF LABORATORY 

FINDINGS, DESIGNED FOR THE USE OF STUDENTS 

AND PRACTITIONERS OF MEDICINE 



BY 

CHARLES C. BASS, M.D. 

\i 

PROFESSOR OF EXPERIMENTAL MEDICINE AND DIRECTOR OF THE LABORATORIES OF CLINICAL 
MEDICINE, THE COLLEGE OF MEDICINE, TULANE UNIVERSITY OF LOUISIANA 

AND 

FOSTER M. JOHNS, M.D. 

ASSISTANT PROFESSOR OF MEDICINE IN THE LABORATORIES OF CLINICAL MEDICINE, THE COLLEGE 
OF MEDICINE, TULANE UNIVERSITY OF LOUISIANA 



ILLUSTRATED WITH 133 BLACK AND WHITE TEXTUAL FIGURES 
AND 19 PLATES IN COLORS 



SECOND EDITION, REVISED 









NEW YORK 
REBMAN COMPANY 



^ 






^?> 



Copyright, 1920, by 
CHARLES C. BASS, M.D. 

• AND 

FOSTER M. JOHNS, M.D. 



m 1 3 192U 



PRINTED IN AMERICA 



©CI.A566545 

! 



PREFACE TO SECOND EDITION 

The entire first edition was so quickly exhausted that 
we feel confirmed in our former opinion that there is great 
need for just such a simple laboratory guide as this was in- 
tended to be. We are indeed grateful for the reception it 
has had. Its adoption and increased use in other schools 
has been especially gratifying. Judging from the useful 
purpose the book has served our students, we look for wider 
use by students — both graduate and undergraduate. 

In the second edition we have endeavored to correct such 
errors as have been discovered in the first edition and to 
bring it up to date in every way. Additions have been made 
wherever necessary, but we have not departed from our 
former aim of putting the essential facts in the shortest and 
clearest form possible. No radical changes have been made. 

We appreciate the kindness of those who have pointed 
cut errors in the first edition and made many valuable 
suggestions. 

The Authors. 



m 



PREFACE TO FIRST EDITION 

Several years of teaching Clinical Laboratory Diag- 
nosis, making laboratory examinations and often interpret- 
ing the findings, has forcibly impressed us with the need of 
such a book as this is intended to be. 

We take up only those laboratory examinations that are 
useful in ordinary, every-day practice and do not include 
tests that are seldom used or that are practical only for 
special laboratory workers. 

Our plan throughout the book is to give only one method 
of making a test or examination. We select what we con- 
sider the best, simplest, most practical method of making 
each test and do not sacrifice space or clearness by giving 
several tests or methods for obtaining the same information. 
All apparatus employed are specified, usually illustrated, 
and when thought to be advantageous, a source of supply 
considered reliable is given. Reagents are all specified, the 
formulae given, and an economical and practical source of 
supply is stated when thought advantageous. 

Some of the technic is original. Many of the "short 
cuts" and simplified steps are our own personal adaptation 
of the work of others. We wish to give full credit here and 
to state that all we know of laboratory diagnosis or any other 
subject has been learned either directly or indirectly from 
the work of others. All the illustrations and colored plates 
are original. They are accurately drawn and colored, fre- 
quently with the aid of the camera lucida. It often occurs 
that a thing can be illustrated better by pictures than by 
written description. Written descriptions are sometimes too 
technical to be easily understood or too long for convenience. 
A single picture often shows as much as can be described 



VI PREFACE 

on a page or more of type. The picture can be read at a 
glance, while the page of print requires minutes to read. 

In an appendix we give a list of the necessary material 
and apparatus required for the laboratory diagnosis de- 
scribed in the text. To do all ordinary laboratory tests one 
requires only a very limited amount of material and appara- 
tus, and a knowledge of how to use them. We are not ad- 
vertising for anybody, but wherever deemed advisable we 
have not hesitated to recommend apparatus or material of 
a particular make or from a source considered reliable. 

In the text we give, as fully as space will permit, the 
interpretation and relative value of the different findings. 
It would, of course, be impossible to discuss thoroughly in 
a work of this kind every possible indication of all the tests. 

Though the book is intended primarily for students and 
those doing only the ordinary every-day clinical laboratory 
work, it is believed that laboratory specialists may also find 
in it some practical suggestions of value to them. 

We wish to express our appreciation of suggestions 
made by many friends and associates, and the opportunities 
that our positions in the Tulane College of Medicine have 
furnished which have permitted the experience and ob- 
servations on which this book is largely based. 

The Authors. 



CONTENTS 

CHAPTER I 

PAGE 

Use and Care of the Microscope 1 

Selection of a microscope — Correct position of the 
microscope — Source of light — Adjustment of the mirror — 
The Abbe condenser — Focussing — Oil immersion lenses. 



CHAPTER II 

Beood 12 

Obtaining blood for all microscopic examinations — Mak- 
ing blood spread for differential leucocyte- count, examina- 
tion for malaria plasmodia, looking for abnormal cells, 
making typhoid agglutination test, etc. — Staining blood 
slides with Wright's stain — Description of the leucocytes 
found in normal blood — Method of making a differential 
leucocyte count — Interpretation of variation in the pro- 
portion of the normal leucocytes in the blood — Description 
of abnormal or pathological leucocytes — Interpretation of 
the presence of pathological leucocytes — Total leucocyte 
counts — Special apparatus and material required — Clean- 
ing the pipette and making the dilution — Making the prep- 
aration — Counting the cells — Interpretation* of total leu- 
cocyte counts — Counting the erythrocytes — Estimation of 
the hemoglobin — Color index — Interpretation of number 
of erythrocytes, hemoglobin per cent, and color index — 
Pathological erythrocytes — Interpretation of pathological 
erythrocytes. 

CHAPTER III 

Malaria 54 

Obtaining blood and making preparations for examina- 
tion for malaria plasmodia — Making the examination and 
recognition of plasmodia — Description of malaria plas- 
modia — Differentiation of malaria plasmodia — Interpreta- 
tion of examination of the blood for malaria. 



Vlll CONTENTS 

CHAPTER IV 

PAGE 

Typhoid Agglutination Test 65 

Technic — Interpretation of the typhoid agglutination 
test. 

CHAPTER V 

Urine 70 

Collection of specimens for examination — Specific grav- 
ity — Test of the reaction — Test for albumin — Test for 
sugar — Test for indican — Test for acetone — Preparation 
of specimens for microscopic examination — Method of ex- 
amination — Diagnosis of microscopic objects commonly 
found in urine — Interpretation of urine examinations. 

CHAPTER VI 

Gastric Contents 87 

Obtaining material to be tested — Test for free HC1 and 
total acidity — Other examinations — Interpretation of the 
findings in gastric contents. 

CHAPTER VII 

Feces ; . 90 

Examination for intestinal parasite ova and larvae — 
Collection of specimens — Making the preparation for 
examination — Concentration of ova by means of the centri- 
fuge — Method of examining a slide preparation — Unci- 
naria americana ova — Ascaris lumbricoides ova — Trichuria 
trichuris ova — Oxyuris vermicularis ova — Hymenolepis 
nana ova — Tenia saginata and tenia solium ova — Larvae 
of uncinaria and strongyloides — Examination for amebas in 
amebic dysentery — Collection of specimens for examina- 
tion — Examination of unstained material — Technic of 
staining am.ebae and examination of stained specimens — 
Differentiation of pathogenic from non-pathogenic amebae 
— Interpretation — Test for occult blood — Technic of test 
— Interpretation. 

CHAPTER VIII 

Pus and Exudates Generally 108 

General remarks — Making the preparation and stain- 
ing — Application in practice. 

CHAPTER IX 

Sputum 112 

Examination for tubercle bacilli — Collecting specimens 
— Making the preparation — Staining — Description of 
tubercle bacilli — Interpretation. 



CONTENTS IX 

CHAPTER X 

PAGE 

Leprosy 120 

Technic of obtaining material and making preparations 
for examination — Staining — Appearance of lepra bacilli — 
Interpretation. 

CHAPTER XI 

Spinal Fluid 122 

Meningitis — Obtaining material and making preparation 
— Meningococci — Pneumococci — Tubercle bacilli — Other 
bacteria — Cells present — Test for globulin increase — Inter- 
pretation. 

CHAPTER XII 
Diphtheria 127 

Principles of laboratory diagnosis of diphtheria — Ma- 
terial required — Making the culture — Incubation — Exam- 
ination of culture — Making preparations for examination 
■ — Staining — Description of diphtheria bacilli — Interpreta- 
tion. 

CHAPTER XIII 

Gonorrhoea 136 

Obtaining material and making preparation — Carbol- 
fuchsin and methylene blue stain — Gram's staining method 
■ — Appearance of gonococci — Interpretation. 

CHAPTER XIV 
Syphilis 141 

Examination for Treponema pallida — Obtaining material 
and making preparation for examination with the darkfield 
condenser — Examination with the darkfield condenser — 
India ink preparation — Differentiation between Treponema 
pallida and other spirochetes — Gland puncture — Interpre- 
tation — Wassermann serum test — Materials required — Ap- 
paratus required — Method of making the test — Control — 
Test — Interpretation. 

Appendix 163 

Complete list of apparatus and material required. 

Index 169 



LIST OF ILLUSTRATIONS 

FIG. PAGE 

1 — Bausch & Lomb microscope 2 

2 — Spencer "No. 44H" microscope 3 

3 — Proper position at the microscope 4 

4 — Learning to look in the microscope with both eyes open. . 5 

5 — Leitz microscope with Mazda lamp 5 

6 — Improper use of concave mirror with Abbe condenser. ... 6 

7 — Abbe condenser , 7 

8 — Abbe condenser in proper position 8 

9 — Abbe condenser too low 9 

10 — Photomicrographs of the same field, showing effect of 

proper and improper illumination 10 

11 — A good blood sticker 12 

12 — Squeezing finger when sticking to lessen pain sense 13 

13 — Squeezing ear lobe 13 

14 — First step in obtaining blood from yourself after sticking 

the finger 14 

15 — Constricting patient's finger after having made the punc- 
ture ; 14 

16 — Squeezing out the blood. 14 

17 — Second step in obtaining blood from the finger 15 

18 — Squeezing blood from the patient's finger 15 

19 — Taking up blood from the patient's finger 16 

20 — Taking up blood on slide from your own finger 16 

21 — Taking up blood on slide from ear lobe of patient. . . 17 
22 — A good general purpose blood spread labeled with an 

ordinary lead pencil 18 

23 — Proper pose of hand and fingers to receive slide with blood 

on it 19 

24 — Slide with drop of blood on it held in proper position for 

spreading 19 

25 — Slides held in proper position preparatory to spreading 

blood 19 

26 — A. Indicating how blood collects beneath the spreader 

slide when the latter is held at proper angle 20 

B. Indicating how blood collects in front of end of the 
spreader slide when the latter is held at too great an 

angle 20 

27 — Indicating angle at which slides should be held and direc- 
tion in which the spreader slide should be moved in mak- 
ing blood spread 21 

x 



CONTENTS XI 
FIG. PAGE 

28 — The blood has been spread by pushing (not pulling) the 

spreader slide quickly , . 21 

29 — Diluting Wright's stain on slide , 23 

30 — Indicating manner of crossing and recrossing blood film 

in making differential leucocyte counts 27 

31 — Looking in the microscope with the left eye in making dif- 
ferential leucocyte counts 29 

32 — Diluting pipette 34 

33 — Bass counting chamber 34 

31 — Counting chamber. Cross section 35 

35 — Bass ruling 36 

36 — Taking up blood into the diluting pipette from the pa- 
tient's finger 37 

37 — Filling diluting pipette with diluting fluid 38 

38 — Revolving pipette to mix contents 38 

39 — Pipette containing diluted blood 39 

10 — Cleaning cover-glass 10 

11 — Counting chamber resting face downward against the foot 

of the microscope after it has been cleaned 10 

42 — Squeezing out diluted blood upon the counting plate 

(Tiirck's counting chamber) 41 

43 — First step in placing cover-glass upon the counting 

chamber 41 

11 — Second step 42 

45— Third step 42 

46— Fourth step 43 

47— Fifth step 43 

48 — Looking at distribution of cells upon ruled plate 44 

49 — Even distribution of cells upon the ruled plate 45 

50 — Uneven distribution of cells upon the ruled plate 45 

51 — Order in which the erythrocytes in the sixteen small 

squares in one large square should be counted 47 

52 — The twelve cells to be counted in this small square are 

shaded 48 

53 — Spreading the drop of water over the blood to dissolve it 

in making typhoid agglutination test 66 

54 — Tilting slide back and forth to facilitate mixing and to 

hasten agglutination 66 

55 — A series of agglutination tests 67 

56 — Looking at typhoid agglutination test 68 

57 — Looking at typhoid agglutination test at night by the 

light of a match 69 

58 — Testing specific gravity with urinometer 71 

59 — Boiling urine in test for albumin 71 

60 — Adding acetic acid to urine from a drop bottle in testing 

for albumin 72 



Xll LIST OF ILLUSTRATIONS 

FIG. PAGE 

61 — Looking for faint cloud in test for albumin 73 

62 — Cloud due to albumin in urine as seen in the proper light 

against a dark background 73 

63 — Filling tube with urine to a depth of 2% inches in quanti- 
tative test for albumin 74 

64 — Preparing fresh Fehling's solution in testing for sugar. . 75 
65 — Boiling Fehling's solution and urine in testing for sugar . . 76 
66 — Adding urine from 1 cc. graduated pipette in quantitative 

test for sugar 77 

67 — Electric centrifuge with Cornell shields 79 

68 — Box guard around electric centrifuge 79 

69 — Hand centrifuge with Cornell shields 80 

70 — Filling balance tube to height of column of urine 81 

71 — Pouring out sediment and spreading on slide with mouth 

of the tube 81 

72 — Proper spread of urine sediment 82 

73 — Proper bottle containing sufficient formed feces for exam- 
ination 91 

74 — Stirring feces in a couple of drops of water on a slide to 

make properly diluted preparation 91 

75 — Proper spread of diluted feces showing also how print 

may be just read through it 92 

76 — Photomicrograph of feces prepared by diluting in the 

ordinary way and showing two tapeworm ova 93 

77 — Photomicrograph of the same specimen of feces prepared 

by centrifuging 94 

78 — Solution of feces made in the bottle in which it was 

brought to the laboratory 94 

79 — Straining diluted feces into centrifuge tube 95 

80 — Diluted feces after centrifuging 95 

81 — Pouring out and spreading sediment from feces on slide 

with lip of centrifuge tube 96 

82 — Diagram indicating proper method of examining entire 

preparation 96 

83 — Photomicrographs of different ova all taken with the same 

magnification for comparison 97 

84 — Photomicrograph of ovum of tenia saginata enclosed 

within its vitelline membrane 100 

85 — Photomicrograph of hookworm ovum and larva and ovum 

of trichuria 101 

86 — Photomicrograph of anterior end of hookworm larva 

under high magnification 101 

87 — Change of shape and position of an endameba during in- 
tervals of a few seconds each by ameboid motion 105 

88 — Drawing illustrating morphological classification of 

bacteria 109 



LIST OF ILLUSTRATIONS Xlll 

FIG. PAGE 

89 — Proper spread of pus or similar material to be stained 

and examined 110 

90 — Thoroughly draining sediment in bottom of centrifuge 

tube 110 

91- — Picking out favorable material from sputum in a Petri 

dish 114. 

92 — Touching the heated slide to the back of the hand to avoid 
getting it hot enough to damage the film of pus when 

fixing with heat 115 

93 — Proper way to apply stain to a slide 116 

94 — Heating slide in staining with carbol fuchsin 117 

95 — Decolorizing with sulphuric acid solution 118 

96^Scraping. leprous lesion 120 

97 — Introducing the needle in making lumbar puncture with 

patient in sitting position 122 

98 — The spinal fluid is allowed to drop directly into the 

centrifuge tube 123 

99 — Bass diphtheria culture tube 127 

100 — Removing cotton plug from culture tube preparatory to 

inoculating with the swab 128 

101 — Inoculating culture media by rubbing swab over surface. . 129 
102 — Sterilizing the platinum loop in flame of Bunsen burner. . 132 
103 — Removing cotton plug, sterile platinum loop held in hand . 132 
104 — Scraping the surface of the culture with the platinum 

loop to obtain bacteria for examination 133 

105 — Proper long thin streak of material from the culture to 

be stained and examined 133 

106 — Decolorizing with alcohol 139 

107 — Scraping chancre 142 

108 — Best form of darkfield condenser 143 

109 — Diagram showing path of rays through a darkfield con- 
denser, and a /I2 inch oil immersion lens fitted with 

funnel stop 143 

110 — Gas filled Mazda lamp for dark-ground illumination. . . . 144 
111 — Illustration of the position of the funnel stop placed in 
the lens case of M2 inch oil immersion lens for darkfield 

work 145 

112 — Photomicrograph of Treponema pallida in "chancre 

juice," as seen with the darkfield microscope 146 

113 — Proper India ink preparation 146 

114 — Photomicrographs of India ink preparations containing 

spirochetes 147 

115 — Etherizing guinea-pig; preparing to draw blood 150 

116 — Introducing the needle while steadying the parts 150 

117 — Drawing blood from guinea-pig 151 



XIV LIST OF ILLUSTKATIOXS 

FIG. PAGE 

118 — Burroughs-Wellcome all- glass syringe with platino-irid- 

ium needle attached 152 

119 — Drawing blood from patient 152 

120 — Further distention of veins by clinching the fist 153 

121 — Squeezing the arm to further distend the veins 153 

122 — Introducing the needle 154* 

123— Drawing the blood 154 

124 — Making pressure over puncture to prevent bleeding under 

skin 154* 

125— Wood test-tube block 2x4x8 inches 155 

126 — Small electric incubator suitable for complement fixation 

tests on a small scale 156 

127 — Showing arrangement of tubes in rack and use of 1 c.c. 

pipette in measuring serum, antigen, etc., into tubes. . 157 
128 — Proper method of washing pipette with saline solution 

from a water bottle equipped with tube and pinch-cock. 158 
129 — Scheme showing distribution of serum, antigen, etc., in 

the control tubes and the test-tubes 159 

130 — Arrangement of work table where electric light is used, 

showing most of the material and apparatus required 

in ordinary microscopic work 164 

131 — Micro burner, much better than the larger Bunsen burner 165 

132- — Proper drop bottle for stains, reagents, etc 167 

133 — Two kinds of improper drop bottles 168 



LIST OF PLATES 

PLATE PAGE 

I — A. Small mononuclear leucocytes 

B. Large mononuclear leucocytes 

C. Polymorphonuclear neutrophilic leucocytes 24 / 

II — A. Polymorphonuclear eosinophilic leucocytes 

B. Polymorphonuclear basophilic leucocytes 

C. Relative sizes of the normal blood elements 24 v 

III — Neutrophils — Eosinophiles — Basophiles — Mature 

cells— Myelocytes 32 ' 

IV — Tallquist hemoglobin scale 49 

V — Abnormal erythrocytes : Anisocytosis — Poikilocytes — 
Megaloblasts — Normoblasts- — Granular or stippled 

erythrocytes — Polychromatophilia 53 " 

VI — Estivo-autumnal malaria plasmodia 55 / 

VII — Tertian malaria plasmodia 55 

VIII — Quartan malaria plasmodia 55" 

IX — Chemical tests of urine 71 v 

X — Microscopical findings in urine 83 

XI — Test of gastric juice for free HC1 and total acidity. . 87 ^ 

XII — Intestinal parasite ova and larvae 97 K 

XIII — A and B. Bloody mucus in amebic dysentery 

C. Preparation from pyorrhea lesion 103 

XIV— Test for occult blood 105 

XV — A and B. Tubercle bacilli in sputum 

C. Lepra bacilli 

D. Pneumococci 118 ,. 

XVI — Diplococcus intracellularis — Diplococcus pneumoniae 

— Bacillus influenzae — Bacillus tuberculosis 124 ' 

XVII — Diphtheria bacilli from different cases suggesting 

variation in morphology 133 l 

XVIII — A and B. Pus in acute gonorrhoea 

C. Pus from case of acute non-specific urethritis 

D. Pus obtained from prostate and seminal vesicles by 
massage in case of chronic gonorrhoea 139 ^ 

XIX — Modified complement fixation test for syphilis 159 



PRACTICAL CLINICAL LABORATORY 
DIAGNOSIS 

CHAPTER I 

USE AXD CAKE OF THE MICROSCOPE 

The microscope is used in a large part of the examina- 
tions made in the clinical laboratory. Therefore it is im- 
portant to have a good microscope and to know how to use 
and care for it. The best microscope will be of little service 
to one who does not know how to use a microscope properly. 

Selection of a microscope. — It so often occurs that those 
beginning laboratory work get useless and improper micro- 
scope equipment that we give here a selection of microscopes 
of the three standard makes in common use in this country 
best suited for the work described in this book. The German 
made Leitz microscope is a somewhat higher grade instru- 
ment than the American made Bausch & Lomb, or the 
Spencer, but costs considerably more on account of import 
duty imposed. It is questionable whether the additional ex- 
pense is warranted when the instrument is purchased for 
routine clinical laboratory work only. Either of the other 
two serves the purpose perfectly, though we have felt a cer- 
tain preference for the Bausch & Lomb. No serious mis- 
take will be made, however, in selecting any one of the three 
specified below. 

Leitz model "II L," with objectives No. 3 (16 mm.), 
No. 6 (4 mm.) and 1 /i2 // (1.8 mm.) oil immersion of N. A. 
1.30; Ocular IV (10X) ; triple nosepiece; Abbe condenser 
of 1.20 X. A. with condenser iris diaphragm in substage 
(Fig. 5). E. Leitz, 30 East 18th Street, New York. 

Bausch & Lomb "F F S," with objectives 16 mm., 4 

1 



2 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 




Fig< l —Bausch & Lomb microscope described on page 1. Shown with 
mechanical stage attached. 



USE AND CARE OF THE MICROSCOPE 



3 



mm. and 1.9 mm. oil immersion; ocular lOx; triple nose- 
piece; Abbe condenser of 1.20 N. A. with condenser iris 
diaphragm (only) in substage (Fig. 1). Bausch & Lomb 
Optical Co., Rochester, New York. 




Fig. 2. — Spencer "No. 44-H." Shown without hiechanical stage. 



Spencer "No. 44-H," with objectives 16 mm., 4 mm. and 
1.8 mm. oil immersion; ocular lOx; triple nosepiece; Abbe 
condenser of N. A. 1.20 with condenser iris diaphragm 
(only) in substage (Fig. 2). Spencer Lens Co., Buffalo, 
New York. 

A mechanical stage (Fig. 1) is essential for best work, 



4 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 



and one made by the same manufacturer and suited to the 
particular microscope stand selected, should be purchased 
with the microscope. 

If darkfield work is to be done (and it should be, if 
diagnosis of 'suspected syphilitic lesions is to be made) a 
darkfield condenser (Fig. 108) to fit in the substage of 
the particular microscope and a suitable funnel stop (Fig. 




Fig. 3. — Proper position at the microscope. Both eyes open. 

Ill) for the oil immersion lens should be purchased at the 
same time. If ordered later give the manufacturers the fac- 
tory number and exact description of your microscope. 

Correct position of the microscope. — Do not tilt your 
microscope stand. Sit up straight, close to the table, having 
the base of the microscope near the edge of the table 
(Fig. 3). Keep both eyes open. To learn to do this it is 
best to first turn the head to one side considerably (Fig. 4) 
and to gradually get it back straight. A few hours' prac- 
tice is usually sufficient to learn to keep both eyes open 



USE AND CARE OF THE MICROSCOPE 




Fig. 4. — Learning to look in the microscope with both eyes open. 
The head is turned to one side. 




Fig. 5. — A good light (Mazda lamp, 2.> watt) and light-holder in proper position. 
Leitz microscope described on page 1, also shown with mechanical stage 
attached. 



6 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

while looking in the microscope with one. Learn to look in 
the microscope with either eye. 




and when using the low power lens (16 mm.)- 

Source of light.— A window on the north side of the 
room is best. Where electric lights are available they give 
the most uniform light and have the further advantage of 



USE AND CARE OF THE MICROSCOPE 7 

being usable any time of day or night. A mazda lamp with 
round frosted globe is best. If some such lamp-holder as 
shown in Fig. 5 is used so the light can be placed near the 
microscope, a 25 watt mazda lamp will give sufficient light, 
otherwise use a 50 watt lamp. 

Adjustment of the mirror. — The mirror has one concave 
and one plane surface. When used without the Abbe con- 
denser (which is seldom done in our work), the concave sur- 
face gives the strongest light by concentrating the rays, 
while the plane surface gives the least light. These condi- 
tions are reversed (Fig. 6) when the Abbe condenser is 
being used. The plane surface 
should be used, therefore, practi- 
cally all the time. The mirror 
must be adjusted so that the best 
light is directed through the ob- 
ject. This is determined by tilt- 
ing the mirror from side to side 
while looking in the microscope. 

Xote the point at which the Fig. 7. — Abbe condenser re- 

. . , . moved from the sub-stage. 

brightest light IS Obtained. lhe This can easily be slipped out 

. -1 j • ■ -1 of its carrier. The iris dia- 

mirror must be readjusted every phragm is opened or closed 

time the micrOSCOpe is moved. by the lever shown at the left 

r lower side of the picture. 

The Abbe condenser. — The 
Abbe condenser (Fig. 7) serves the purpose of concen- 
trating the light rays and directing them at a different 
angle through the object. It is carried in a slip sleeve in 
the substage. It frequently gets pushed too low in this 
sleeve and this is a great source of poor illumination. 
Refer to Figs. 8 and 9 and note that proper illumina- 
tion is obtained by having the surface of the condenser 
approximately as high as the surface of the stage of the 
microscope. Examine your Abbe condenser and learn 
how it can be moved up and down, and look out for its 
getting out of place. There is an iris diaphragm under 
the condenser with which the amount of light must be regu- 
lated. It is strange how long it takes students to learn to 




8 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 



appreciate the necessity of regulating the light for different 
objects. In practice the light should be adjusted and regu- 
lated for practically every slide examined (Fig. 10), and 




Yia-, 8. — Illustrating manner in which light rays reflected from the flat mirror 
b are focussed upon the object by the Abbe condenser in proper position. 

frequently many times in examining a single specimen. 
Learn this now. Generally speaking, the higher the magni- 
fication the more light required. With low power lenses it 



USE AND CARE OF THE MICROSCOPE 



9 




Fig< 9.-Compare with Fig. 8 and note the ioss of light upon the object, where 

it is des red, resulting from the Abbe condenser being too low. It often 
happens that the condenser is slipped down in the sleeve, which carries it 
and is not noticed and the microscope is used at this great disadvantage 
for some time. Watch out for this. 

is necessary to reduce the light. Also the more color an 
object has the more light is required. Hyaline and un- 
stained objects require less light. 



10 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 




Fig. 10. — Photomicrographs of the same field. A. Too much light. Note that 
the hookworm egg is poorly shown and other objects cannot be seen. 
B. Proper amount of light obtained by reducing size of the opening in 
the iris diaphragm. 

Focussing. — Place the part of the object to be examined 
approximately in the center of the field which can be guessed 
by its position over the Abbe condenser. With the coarse 
adjustment bring the tip of the objective near the object; 
then look in the microscope and rack upward until the ob- 
ject appears. Then focus with the fine adjustment. Ma- 
nipulate the fine adjustment with the left hand and the 
mechanical stage with the right (Fig. 3). 

Use of oil immersion lenses. — Only the best cedar oil 
"for immersion" should be used. It gets gummy and col- 
ored upon long exposure to light and air. Don't use such 
oil. Don't let dust get into it. Put a small drop of oil on 
the specimen over the place selected to examine. Run the 
tip of the oil immersion lens down until it touches the oil. 
Now focus as with other objectives. After use the oil must 
be cleaned off. Wipe the lens carefully with a soft linen 
cloth. A handkerchief is good for this purpose. Never let 
oil dry on the lens. If you do, remove it with a cloth 
moistened with xylol. 



USE AND CARE OF THE MICROSCOPE 11 

Cleaning the lenses. — The exposed surfaces of oculars, 
objectives, condensers and mirrors are likely to have dust 
particles collect upon them at any time. It is best to wipe 
them off with a soft linen cloth (handkerchief) just before 
use each time. Keep the ocular and objectives attached to 
the microscope all the time, and there is little danger of dust 
getting on the back lenses of the objectives and lower lens 
of the ocular. They require cleaning only occasionally. 
Sometimes the inner surfaces of the lenses in the ocular 
require cleaning due to a film that forms on them. This you 
can do. Objectives should be sent to the makers every three 
to five years or oftener, if necessary, to be cleaned. Don't 
try to clean them inside. You should clean the back lens 
of the objectives occasionally by wiping with a suitable piece 
of cloth wrapped on a small wooden stick or applicator, or 
with the corner of the cloth rolled into proper shape. 

Care of the microscope stand. — It is better to keep the 
microscope assembled and on the table ready for use. A 
bell jar is a good cover to protect it from dust. Wipe it 
occasionally with a slightly oily cloth. Wipe off any 
gummy oil on the bearings and oil them a little with soft 
tallow or white vaseline. If gummed too badly, remove 
by wiping the bearings only with cloth moistened with 
xylol. You can put a little oil in the bearings of the 
coarse adjustment. If anything gets wrong with the fine 
adjustment send the microscope to the makers for repairs. 
Don't try to repair it yourself. 

Clean and oil the mechanical stage in the same way that 
you do the stand. Be sure to put the mechanical stage 
on so that it does not bind and drag on the stage of the 
microscope. 



CHAPTER II 

BLOOD 

Obtaining blood for all microscope examinations. — Some 
kind of a "blood sticker" is required. A good and con- 
venient one may be made of a straight Hagedorn needle 
No. 6. The point of most of them is too long and narrow. 



i 



Fig. 11. — A good "blood sticker." A properly sharpened straight 
Hagedorn needle No. 6, in 2 dram homo vial. 

Grind it to a proper short angle. Stick the eye end of the 
needle into a cork stopper and insert it into a two dram 
homo vial to carry it in (Fig. 11). 

The side of the first phalanx of one of the fingers is a 
convenient place to draw blood from. This is especially 
true when taking blood from yourself. One should learn to 
take blood and make the spreads with his own blood. It is 
(sometimes) more convenient to draw blood from the ear- 
lobe of patients. The principles of the technic are the same. 

Wipe the skin dry at the site it is intended to stick. If 
the skin is dirty, wash it, but it is not necessary nor desirable 
to sterilize with antiseptics, except, perhaps, in special in- 
stances. Squeeze the place to be stuck between the thumb 
and finger and make a quick stick with the needle while 
squeezing. The pressure lessens pain sense. Most workers 
can govern the depth and accuracy of the stick better if they 
steady the hands by letting them rest upon a table or other 
surface when sticking a finger (Fig. 12), or against the side 
of the face and neck of the patient when sticking the ear 

12 



BLOOD 



13 




Fig. 12. — Squeezing finger when sticking to lessen pain sense. 




Fig. 13. — Squeezing the ear-lobe and steadying the hand, against the face- 
when making the stick. 

(Fig. 13). The stick should not be deep enough to cause 
blood to flow without proper manipulation of the surround- 
ing tissue. It should be made so quickly and well that the 
patient hardly knows when it is done. 



14 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 




Fig. 14. — First step in ob- 
taining blood from your- 
self after sticking the 
finger. The blood is pre- 
vented from running back 
by pressure made around 
the finger by squeezing it 
in this manner. 




Fig. 15. — Constricting patient's finger after hav- 
ing made the puncture, the first step in 
squeezing out blood. 




Fig. 10.— Squeezing out the blood. Note that the pressure is not made 
close to the puncture. 



BLOOD 



15 



To squeeze out the blood use both hands. Try to en- 
tirely surround the end of the fingers (Figs. 14 and 15) or 
ear-lobe (Fig. 16) with your fingers so that the blood can- 
not escape back. Your fingers should not be nearer than 
from one-fourth to one-half inch of the stick (Figs. 17 




Fig. 17. — Second step in obtaining blood from one's own finger. Note that pres- 
sure is not made near the site of the puncture. The pressing fingers of the 
right hand are held nearly parallel to the bleeding finger, and pressure is 
made in this manner while the blood is prevented from returning by the con- 
striction made by the fingers of the left hand. 




Fig. 18. — Squeezing blood from patient's finger. Note that pressure is not 
made close to the puncture. 



and 18). If you hold and press too close to the stick, little 
blood will be obtained. Hard squeezing dilutes the blood 
with tissue juices and must be avoided. Squeeze out the 
proper amount with which to make one spread. About 
one-fourth drop is the proper amount. Stop the pressure 



16 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 




Fig. 19. — Taking up blood from patient's finger, 




Fig. 20. — Taking up blood on slide from your own finger. The edge of the slide 
is first steadied against your left forefinger. 



and no more blood will flow if the stick was of the proper 
size and depth. If the skin is dry, the blood will stand 
up properly as a small round mass, but if it is not dry it 
will spread out on the skin. The proper amount of blood is 



BLOOD 17 

taken up on a clean slide by touching the slide to it. Do 
not let the slide touch the bleeding finger. It is best for 
most operators to steady the hand which holds the slide 
against the table (Fig. 19), or the other hand (Fig. 20), 
or the face (Fig. 21) of the patient when taking blood 
from the ear, before trying to touch the slide to the drop 
of blood. The blood is now spread out upon the slide and 
then another proper amount of blood is squeezed out and 



Fig. 21. — Taking up blood on slide from ear-lobe of patient. This picture 
erroneously shows the blood about to be taken up on the wrong end of 
the slide. It should be taken on the lower end. 



spread upon another slide in the same way. It is best to 
make at least two good preparations, and preferably three, 
in every case. If a proper stick is properly manipulated, 
twenty-five to fifty preparations can be made from it if 
desired. 

Making blood spread for differential leucocyte count, 
examination for malaria plasmodia, looking for abnormal 
cells, making typhoid agglutination test, etc. — The ad van- 



18 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

tages of well made blood spreads are sufficient to justify the 
effort necessary to learn to make them. There are compara- 
tively few who can make good ones. A general purpose 
blood spread should have both thick and thin portions, and 
there should be no dragging of the larger cells. The blood 
should be spread upon the middle third or a little more of 
the slide and should not reach to either end (Fig. 22). 




Fig. 22. — A good general purpose blood spread labeled with an ordinary 

lead pencil. 



Have the slides clean and free from chemicals. When 
not soiled with oily or gummy material and stains, they may 
be washed in water and wiped dry with a towel or cloth. 
Hold by the edges when wiping. Oily or stained slides may 
be cleaned by boiling them in water to which has been added 
a little washing powder or soap, and subsequently rinsing 
well. 

Take up the blood on the slide at about three-fourths 
of an inch from one end. The slide may be placed upon 
the table while spreading the blood, or it may be held in the 
hand. If held in the hand, which is preferable, first hold the 
hand in the position shown in Fig. 23, ready to receive the 
slide. Place the end of the slide farthest from the blood drop, 
between the thumb and finger (Fig. 24), and bring up the 
little finger to support the other end, or lay it down on the 
table before you. Whether the slide is lying on the table or 



BLOOD 



19 





Fig. 23. 



Fig. 24. 



Fig. 23. — Proper pose of hand and fingers to receive slide with blood on it. 
Fig. 24. — Slide with drop of blood on it held in proper position for spreading. 




.Fig. 25. — Slides held in proper position preparatory to spreading blood. 



20 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

held in the hand, the blood is spread with another slide 
held in the other hand. Place the end of the spreader 
slide just in front of the drop of blood and bring it back 
to it (Fig. 25). Hold it at an angle of about thirty de- 




Fig. 26. — A. Indicating how blood collects beneath the spreader slide when the 
latter is held at proper angle. B. Indicating how blood collects in front 
of the end of the spreader slide when the latter is held at too great an angle. 

gre.es (Fig. 26). The blood spreads toward both edges 
and is all behind the sharp edge of the end of the spreader 
slide. Now, with a rather quick movement, push (Fig. 27) 
the spreader slide toward the end of the lower slide (Fig. 
28) spreading out the blood in a thinner or thicker film, 
according to whether the movement is slow or rapid re- 
spectively. It is desirable to leave the blood on the middle 
third of the slide or a little more. Note (Fig. 22) that 
the last part of the spread is considerably thinner than 
the first part. Such a spread offers fields of any desired 
thickness. One made with too slow a motion resulting in 
a very thin preparation will always show dragging of the 
leucocytes, which alters the differential count. Allow the 
blood to dry. The preparation may be labeled by writing 



BLOOD 



21 



on the blood with a pencil after it has dried. Such slides 
will keep for several days unstained, if not convenient to 
stain them earlier. However, you will generally 
get the best stains on fresh specimens. 




Fig. 27.— Indicating the angle at which slides should be held and the direction in 
which the spreader slide should be moved in making blood spread. 





J 

Fig. 28.— The blood has been spread by pushing (net pulling) the spreader 

slide quickly. 



Staining blood slides with Wright's stain.— There are 
several good methods of staining blood. We prefer Wright's 
stain. It is a good one. Wright's stain is a solution of a 



22 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

mixture of eosinate of methylene blue and eosinate of meth- 
ylene azur, prepared in a certain way described by J. H. 
Wright {Journal A. M. A., Vol. LV, p. 1979), dissolved 
in pure methyl alcohol. The powder may be purchased in 
bulk or in the form of tablets (6 tablets in a bottle) made 
by Burroughs, Wellcome & Co., or the staining solution 
may be purchased ready for use. Much of the latter sold 
is unsatisfactory. 

To make the solution from the powder, dissolve 0.2 gm. 
in 100 c.c. of Merck's methyl alcohol, highest purity. To 
make it from the tablets, pulverize and dissolve 6 tablets 
(contents of one vial) in 75 c.c. of the alcohol. The stain 
improves with age for a week or two. If kept in well-closed 
bottles and out of bright light, it keeps almost indefinitely. 

The technic of staining is as follows : 

1. Flood spread with Wright's stain about one minute. 

2. Dilute with about two to three times as much water. 
Allow to stain five to ten minutes. 

3. Wash, dry and examine with the oil immersion lens. 
The object of the first step is to fix the preparation 

(harden the cells and incidentally to stick the film to the 
slide) and is accomplished by the methyl alcohol with which 
the stain is dissolved. The slide should be placed on a slide- 
rest or staining bar over a waste jar and not held in the 
hand. 

The amount of stain poured on the slide should be the 
minimum that will cover the film well and not evaporate 
sufficiently to throw down a precipitate on the slide in the 
one minute it is allowed to act. In about one minute after 
the stain is put on the slide it begins to change to a purplish 
color, and to throw down a precipitate. Just at this point 
is the proper time to add the water. It is better to watch 
for this than to go by exact time. 

Add the water from the water bottle (Fig. 29), the tube 
from which should hang over the staining bar and jar, and 
it should be just long enough to clear the slide. By noting 
the amount of staining fluid on the slide one can guess when 



BLOOD 



23 



he has added two or three times as much water. It is often 
necessary to vary the quantity of water added somewhat 
with different samples of staining solution. It is also some- 
times necessary to vary the length of time the diluted stain 
is allowed to remain on the slide, with different samples. 




Fig. 29. — Diluting Wright's stain on slide. Slide resting upon the slide-rest 
over a waste jar. Water from water-bottle. 

A good plan when beginning the use of a stain with which 
you are not familiar is to stain several slides different lengths 
of time, say from one to ten minutes, and see which is the 
best. The water used to dilute and to wash with must be 
either distilled or at least of high purity. Rain water is 
usually good. 

After washing the preparation it should be promptly 
dried. First wipe the back of the slide and the front around 



24 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

the spread. Drying may be hastened by fanning the slide 
in the air or by propping it up on end against some con- 
venient object. 

Description of the leucocytes found in normal blood. — 
There are at least five different kinds of leucocytes present 
in normal blood. They are differentiated from each other by 
the size, shape and staining reaction of the nuclei, cytoplasm 
and granules present in the cytoplasm of some of them. The 
classification which we believe most nearly correct, in the 
light of our present information, is as follows: 

A. Small mononuclear leucocytes. (Lymphocytes.) 
These cells (Plate I) have a single round or oval nucleus 
which is frequently indented on one side. They have a 
relatively small amount of cytoplasm. Some have only a 
narrow band surrounding the nucleus, while others have 
very much more. They vary in size from about the diam- 
eter of an erythrocyte to about twice that size. The nu- 
cleus stains a beautiful purple in well-stained specimens 
and the cytoplasm varying shades of deep blue. The nu- 
cleus and cytoplasm are sharply differentiated. In the 
cytoplasm of a good many of the lymphocytes, there are 
from one to several reddish stained granules. They con- 
stitute about 20% to 30% of all the leucocytes in the blood 
of normal adults. 

B. Large mononuclear leucocytes. These cells (Plate I) 
are larger than the lymphocytes and two to three times the 
diameter of erythrocytes. Both the nucleus and cytoplasm 
appear to be less dense than those of the small mononuclear 
leucocytes, and frequently the cytoplasm is not so clearly 
differentiated from the nucleus. The proportion of the 
cytoplasm to nuclear material is much greater than in the 
small mononuclear leucocytes. The nucleus stains purple 
and the cytoplasm varying shades of light blue. In deeply 
stained specimens the cytoplasm of the older cells approaches 
the lavender of the neutrophile, making the differentiation 
depend largely on size and amount of cytoplasm. The nu- 
cleus varies in shape from round or oval to slightly lobu- 
lated. It is often placed more or less concentrically. Some 



late I. 








\. Small Mononuclear Leucocytes 







B. Large Mononuclear Leucocytes. 



U«a l \4ift 




C. Polymorphonuclear Neutrophilic Leucocytes. 



Plate II. 









t>» 



A. Polymorphonuclear Eosinophilic Leucocytes. 



«c 



;*•" 






*>-Vi ft 



* W ■ 






■5*5* 



* ' ■ - v j ' ■** . 



i 



V 



^* 



k ? 



#0 






B. Polymorphonuclear Basophilic Leucocytes. 








C. Relative Sizes of the Normal Blood Elements. 



BLOOD 25 

of the large mononuclear cells contain no granules, but most 
of them do show from a few to many purple-stained gran- 
ules in their cytoplasm. These vary in size between that 
of the granules in neutrophiles and those in eosinophiles. 
The large mononuclear leucocytes constitute about 1% to 
8% of all the leucocytes in the blood of normal adults. 

C. Polymorphonuclear neutrophilic leucocytes. These 
cells (Plate I) are from two to nearly three times the diam- 
eter of erythrocytes. The nucleus stains purple and is 
polymorphous, every one being somewhat different in shape 
from every other one. The outline of the nucleus is very 
irregular, and most of them are more or less lobulated. 
When studied carefully under high magnification the 
nucleus appears to consist of a string or ribbon of nuclear 
material wadded up in the cytoplasm of the cell. Some- 
times the bands connecting the lobes or masses are so very 
thin that they can be made out only with difficulty. In 
other (younger) cells the nucleus is horse-shoe shaped. 

The cytoplasm does not stain, but it contains many fine 
granules which do stain purple, some taking more of the 
red than others. The fact that the cytoplasm of these cells 
had no special affinity for either the basic or the acid stains 
then employed led to their being called neutrophilic or neu- 
trophiles. In speaking of them it is common to call them 
neutrophiles, dropping the other cumbersome part of the 
name. The polymorphonuclear neutrophilic leucocytes con- 
stitute about 60% to 70% of all the leucocytes in the blood 
of normal adults. 

D. Polymorphonuclear eosinophilic leucocytes. These 
(Plate II) average very slightly larger than the neutro- 
philes. Their nuclei resemble very much the nuclei of the 
neutrophiles, but there is possibly greater tendency for the 
nucleus to be divided into distinct lobes (2 or 3). The 
nuclei are often horse-shoe shaped. The cytoplasm does 
not stain, but there are many large granules embedded 
in it, which take the eosin in a mixture containing eosin, 
and the cells are therefore called eosinophilic or eosino- 
philes, dropping the other cumbersome part of the name. 



26 PEACTICAL CLINICAL LABORATORY DIAGNOSIS 

They constitute about 1% to 4% of all the leucocytes in 
the blood of normal adults. 

E. Polymorphonuclear basophilic leucocytes. These 
cells (Plate II) are slightly smaller than the neutrophiles. 
The nucleus stains faintly and the lobes are less distinct. 
It more than half fills the cell. The cytoplasm does not 
stain. There are a variable number of large coarse granules 
which seem to project from the surface. They take the 
basic stains like methylene blue and on account of this 
affinity for basic dyes the cells are called basophilic, or baso- 
philes. The latter term is generally used. These granules, 
as well as a few finer granules sometimes in the cytoplasm, 
are also slightly azurphilic and in specimens well stained 
with Wright's stain their color is a combination of the dark 
blue methylene blue and the purplish azur. The basophiles 
constitute less than 1% of all the leucocytes in the blood 
of normal adults. 

Method of making a differential leucocyte count. — A 
differential leucocyte count is a determination of the rela- 
tive proportion or percentage of the different kinds of 
leucocytes present. The ordinary blood spread described 
above (Fig. 22), stained with Wright's stain, is suitable for 
this purpose. Differential counts are made with the oil im- 
mersion lens. A mechanical stage is an advantage. Begin- 
ners and those not thoroughly familiar with the different 
cells should make their counts on a comparatively thin part 
of the spread where it is not more than one cell thick. After 
more experience, more rapid counting can be done on the 
thicker part of the spread. A good spread has areas, vary- 
ing from too thick to count well, at the thick end, to un- 
necessarily thin, even for the beginner, at the other end. 
It frequently takes a long time for beginners to learn to 
locate proper ground to study. They do not realize the 
extent of the preparation under such high magnification. 
The student should learn early to select the proper part of 
the preparation. The great difference can be best ap- 



BLOOD 



27 



predated by looking over the spread carefully from one 
end to the other. 

Whenever favorable ground is located move toward 
one edge of the spread until the edge is reached (Fig. 30), 
keeping tab of all leucocytes observed. Then move toward 
one end or the other at least the width of the microscope 
field, and then across the spread to the opposite edge. 
In this way cross the spread back and forth, always on new 
ground, until the desired number of cells have been counted. 




Fig. 30. — Indicating manner of crossing and recrossing blood film in making 
differential leucocyte counts. 



No cell is counted twice. All cells passed over must be 
counted. If they cannot be classified, put them down as 
unclassified cells. At first a good many that cannot be 
classified are found, but with experience the student is soon 
able to classify practically all the cells found in normal 
blood. 

In counting, a tally sheet should be provided on which 
to mark down the different cells as they are found. It is 
not necessary to write out the names of the different cells 
as these are familiar terms, which can as well be indicated 
by proper abbreviations. S, may stand for small mono- 
nuclear leucocytes; L, for large mononuclear leucocytes, 
and N, E and B, for polymorphonuclear neutrophilic, 
eosinophilic, and basophilic leucocytes respectively. Like- 
wise U may stand for unclassified leucocytes. A mark is 



28 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

made opposite the proper class for each cell found. For 
convenience in counting up, it is best to tally with the 
mark for every fifth cell of any kind. After a few cells 
have been counted the tally sheet will look something like 
this: 

situ mi i 

Lll 

JVM IU1IU IN BUI 
si 

B 

irlll 

The percentage of each kind of cell can be calculated 
after any number has been counted, but it is much better 
to count exactly one hundred cells, in which case the 
number of each kind of cell counted is also the percentage, 
and no other calculation is necessary. After a little experi- 
ence one learns to stop and add up when nearly one hundred 
cells have been counted. The number required to make 
the one hundred is now found in the specimen and then 
added. 

At first this is slow work, but one should practice to 
acquire speed. After a little time, instead of stopping to 
put down each cell, he learns to carry several cells "in his 
head" before stopping to put them down. This saves 
time. Finally, one should learn to carry the small mono- 
nuclears and neutrophiles "in his head" until nearly one 
hundred have been counted, only stopping to put down the 
other cells which are few in number. When the number of 
cells reaches nearly one hundred they are all put down 
and the tally sheet would look something like this: 



BLOOD 



29 



J* 23 

zm 
jru 

£11 

m 

On adding up it is seen that three cells are required to 
make one hundred. These are. found and added and the 
tally sheet completed. It would look as follows: 

/? 23 23 

£JW, S 

JT U + 2 &S 

£11 + 1 3 

31 I 

Another important short step in making differential 
leucocyte counts is to look in the microscope (Fig. 31) with 



"* 




Fig. 31. — Looking in the microscope with the left eye in making differential 
leucocyte counts. The tally can be plainly seen with the right eye with- 
out moving the head. 



30 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

one (the left) eye and look at the tallying with the other 
(the right) eye, without moving away from the microscope. 
This can be learned with a little practice and has many 
advantages. It is worth learning. With good methods 
and with practice one should be able to count one hundred 
cells in three minutes or less time. 

Somewhat more exact figures can be obtained by count- 
ing several separate hundred cells and taking the average 
of all the counts, but much more depends upon properly 
made spreads than upon counting large numbers of cells. 
No count of the cells on a poor spread in which the larger 
cells have been dragged off, can tell the proportion of the 
cells in the original blood. The student should count several 
separate hundreds on the same spread and on different 
spreads of the same blood and see for himself how much 
they vary. When the count is carefully made on a properly 
made spread and in the part of the spread made before 
there was any dragging of cells whatever, there is very little 
variation indeed. In practice, therefore, a properly made 
count of one hundred cells is sufficient for practical pur- 
poses. 

Interpretation of variation in the proportion of the nor- 
mal leucocytes in the blood. — It should be understood that 
whenever the total number of a given kind of leucocyte is 
increased or decreased the percentage is correspondingly 
increased or decreased accordingly, but the percentage of 
the other cells is decreased or increased though their total 
number may remain exactly the same. Frequently what 
may be a low percentage for a given cell may in fact not 
indicate any actual loss of such cells, but on the contrary 
an increase in other cells, or vice versa. 

The percentage of the different cells in the blood of 
normal adults should be memorized and "at your tongues 
end" at all times. It is: 



BLOOD 31 

S— 20% to 30%. 
L— 1% to 8%. 

X— 60% to 70%. 
E— 1% to 4%. 
B— to 1%. 

In children under five years of age there are usually 
from 40% to 50% small mononuclear leucocytes. Often 
there are even more in very young children. The per cent, 
of neutrophiles is correspondingly less. With increased age 
the proportions gradually change toward those of the cells 
in adult blood which are reached soon after puberty. Most 
women have a little larger per cent, of small mononuclear 
cells and a correspondingly smaller per cent, of neutrophiles. 
It is not at all uncommon to find these cells to be more 
than 30% of all of the leucocytes in the blood of perfectly 
well women. The small mononuclear leucocytes are in- 
creased in those diseases in which there is irritation of the 
lymph tissues of the body, and especially those in which the 
disease is fought or resisted largely by this kind of cell. 
Syphilis and tuberculosis and typhoid fever are* examples 
of such diseases, in all of which there is more or less in- 
crease of the per cent, of the small mononuclear leucocytes 
present. 

The large mononuclear leucocytes vary so much in health 
and also under influences not now recognized that slight 
variations have no special significance. They are greatly 
increased in acute lymphatic leukemia. 

The neutrophiles are the phagocytes of pyogenic bacteria 
and their number in the blood is increased in the presence of 
any disease caused by pyogenic bacteria, provided, of course, 
nature is able to respond to the call and furnish them. 
When pyogenic bacteria are present in a tissue from which 
drainage is poor or impossible and their toxins are ab- 
sorbed, very large numbers of neutrophiles are present; 
but when there is good drainage, and therefore little or no 
absorption of toxin into the system, there is little or no in- 
crease of the neutrophiles. For instance, staphylococci in 



32 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

the appendix or in the peritoneal cavity would give rise to 
a very high neturophile count, while the same bacteria on a 
skin lesion or other surface lesion would give rise to little or 
no increase; gonococci in a joint would give rise to a high 
count, while there occurs little or no increase in simple infec- 
tion of the urethra with the same bacteria; streptococci in 
a middle ear abscess give rise to great increase of neutro- 
phils before drainage is established, but afterward they fall 
to near or quite normal in a few hours. An increase in the 
per cent, of neutrophiles indicates therefore absorption of 
toxin from a pyogenic infection or disease in some part of the 
body, but cannot point out the tissue involved or the par- 
ticular bacteria concerned. All other things being equal, the 
less drainage, the higher the count. A very small focus of 
disease in a tissue from which there is no drainage, like, 
for instance, in bone, under periosteum, in the middle ear, 
etc., may give rise to a high neutrophile count. A high 
neutrophile count indicates, therefore, not only disease proc- 
ess due to pyogenic bacteria, but also that drainage is poor 
or lacking. About ninety-four is the highest neutrophile 
percentage that is often seen. 

The neutrophiles are reduced or at least not increased 
in diseases due to non-pyogenic bacteria, or those not due to 
bacteria of any kind. Of the common pyogenic bacteria 
we may mention pneumococci, streptococci, meningococci, 
staphylococci of almost all kinds, and gonococci. Of the com- 
mon disease-producing organisms that are non-pyogenic may 
be named malaria plasmodia, typhoid bacilli, tubercle bacilli 
(unless in very large numbers), and tetanus bacilli. 

The eosinophiles are usually increased in intestinal para- 
site infections, especially hookworm infection, in which they 
sometimes reach 15% to 20% or more. They are increased 
in trichinosis, sometimes to 75% or more. There is usually 
great increase during and soon following an attack of true 
bronchial asthma. There is slight to considerable increase 
in a variety of parasitic skin diseases. Eosinophilia also 
occurs in most cases of foreign protein poisoning and ana- 
phylaxis. 



Plate III. 







EM 



Basophiles. BM '' : j£gW 



Mature Cells 



Myelocytes. 







Myelocytes. 



The upper half of the picture traces backward the development of the polymor- 
phonuclear leucocytes from myelocytes to mature adult neutrophiles eosinoyhiles and 
bosoyhiles. N-neutrophile; NM-neutrophilic myelocyte; E-eusenoyhile ; EM-euseno- 
philic myelocyte; B-bosophile: MB-bosophilic myelocyte. 



BLOOD 33 

The eosinophiles are decreased by pyogenic infection 
and frequently none are found. An increase of the neutro- 
philes with marked decrease of the eosinophiles is known as 
"Simon's septic factor" and is a very strong indication of 
pyogenic disease or sepsis. 

The basophiles are increased to large proportions in 
some cases of myelogenous leukemia. 

Description of abnormal or pathological leucocytes. — 
The neutrophiles, eosinophiles and basophiles found in the 
normal blood are the descendants of myelocytes which are 
normally present in bone marrow and other blood-making 
tissue of the body. In certain diseases, notably myeloge- 
nous leukemia, myelocytes in large numbers appear in the 
circulating blood. Within certain limitations most of them 
can be grouped under one or the other of the following 
three heads: 

A. Neutrophilic myelocytes. These (Plate III) are 
large cells, having a single round, or oval-shaped nucleus, 
and neutrophilic granules, which are like the granules in 
polymorphonuclear neutrophiles, in the cytoplasm. The 
cytoplasm sometimes takes more or less of the methylene 
blue in Wright's stain. 

B. Eosinophilic myelocytes. These (Plate III) are 
large cells having a single round or oval nucleus, and 
eosinophilic granules which are like the granules in poly- 
morphonuclear eosinophiles, in the cytoplasm. The cyto- 
plasm usually takes more or less of the methylene blue in 
Wright's stain. 

C. Basophilic myelocytes. These (Plate III) are large 
cells having a single round or oval nucleus and basophilic 
granules which are like the granules in polymorphonuclear 
basophiles in the cytoplasm. 

It should be appreciated that the myelocytes are simply 
young cells that have appeared in the circulation before 
they had aged into the polymorphonuclear cells, and there 
must therefore be a good many cells in such a blood that 
are on the border line (Plate III) between them. 



34 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 



In addition to the abnormal leucocytes which fall in one 
or the other of the above groups, there are usually a good 
many others in leukemic blood that cannot be classified. 
They should all be grouped under the head of unclassified 
cells. When making a differential leucocyte count and 
myelocytes are found present, one should add to the usual 
tally sheet a head for each variety, as, for instance, MN, 
ME and MB. 

Interpretation of the presence of pathological leuco- 
cytes. — Myelocytes (approaching the adult polymorphonu- 
clear neutrophile cell type) may occasionally be found in 
small numbers in blood in which there is a considerable 
septic leucocytosis, and sometimes when there is no leuco- 
cytosis. They are generally counted in and considered with 
the neutrophiles. Large numbers are not found except in 
some form of myelogenous leukemia. 

Total leucocyte counts. Special apparatus and material 
required. — The special apparatus required are: A. Dilut- 
ing pipette ( Fig. 32 ) . This should provide for diluting the 
blood one hundred times. B. Counting chamber (Fig. 33). 
The counting chamber devised by one of us (Bass) has 




Fig. 32. — Diluting pipette. 



0.4 cumm 
0.04 •• •• 
0.004 •• 
0.00025- 



r\ 



^/ 



n 




BASS RULING 



Fig. 33. — Bass counting chamber. 



BLOOD 35 

advantages over all others. The chief advantage lies in the 
simplicity of the ruling. This same Bass ruling is now 
supplied on the several different styles of counting chambers. 

<K 

~t f ^ 1— 

c \ c 

Fig. 34. — Counting chamber. Cross section, a. Object slide, b. Counting plate, 
c. Rectangular plates to support cover-glass exactly 0.1 mm. above the count- 
ing plate, d. Special thick cover-glass. 

It consists of a heavy object slide upon which a small oblong 
counting plate is cemented ( Fig. 34 ) . On either side of the 
counting plate there is cemented upon the slide a rectangular 
glass plate so as to leave a gap between the edges of the 
counting plate and the inside edges of these rectangular 
plates. The latter are exactly 0.1 mm. higher than the 
counting plate, so that when a cover-glass is placed over 
both there will be a space of 0.1 mm. between it and the 
counting plate. 

The Bass ruling (Fig. 35) in the middle of the counting 
plate consists of a square of 2 mm. side. This is divided into 
ten rectangles, 0.2 mm. X 2 mm., for counting leucocytes. 
For counting erythrocytes there are superimposed upon the 
ruled space at each corner and in the center five squares of 
0.2 mm. side, each further divided into 16 small squares 
of 0.05 mm. side. The cubic contents of the entire ruled 
space, 0.1 mm. deep, is 0.4 cu, mm. The total cubic contents 
of the five large squares is 0.02 cu. mm. 

In addition to this special apparatus a diluting fluid is 
required. We use Toison's fluid, which permits counting 
both leucocytes and erythrocytes in the same preparation. 
The formula is: 

1? Sodium chloride 1 gm. 

Sodium sulphate 8 gm. 

Glycerine 30 c.c. 

Methyl violet, 5B 0.025 gm. 

Water 160 c.c. 



36 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 



It should be kept in bulk in a tall, narrow bottle and 
whenever a count is to be made, enough of the solution 
should be poured off (into a small bottle) for present use. 
Bo not use directly from stock bottle. Do not return unused 



__ L , 9 

___ . . & 

*•— ————__. — — — — I — — ' 

^ _. «____, 1 1 1 i . I . 

«__ .i.i i ..I » 

— . — — — — ____*„_„___________ -^ : ___._.__ _ _ « 



Fig. 35. — Bass ruling. 

solution to stock bottle. If managed as directed the solution 
usually keeps well, otherwise it may deteriorate from the 
growth of moulds and yeasts and soon be unsatisfactory. 

For counting leucocytes only, a 2% solution of acetic 
acid in water, which destroys the erythrocytes and makes 
the leucocytes easier to see, is useful. It can be made up 
extemporaneously when required for use, or it may be 



BLOOD 37 

poured from a stock bottle when required as advised above 
for Toison's solution. An 0.85% sodium chloride solution 
("Normal saline") is a fairly good diluting fluid when 
the erythrocytes only are to be counted. 

Cleaning the pipette and making the dilution,— The 
pipette must be clean and perfectly dry before use. As- 
suming that it has been used it is cleaned and dried as 
follows: (1) Slip off the rubber tube and blow through 
the tip of the pipette to expel any fluid present. (2) Re- 
place the rubber tube and draw pipette full of water. (3) 




Fig. 36. — Taking up blood into the diluting pipette from the patient's finger. 
Note that the operator's right hand is steadied by touching a finger against 
the finger of the patient. 

Slip off rubber tube and blow water out. (4) Replace 
rubber tube and draw pipette full of alcohol. (5) Slip off 
rubber tube and blow alcohol out. (6) Replace rubber 
tube and draw pipette full of ether. (7) Slip off rubber 
tube and remove ether by slinging the pipette somewhat 
like "shaking down" the mercury in a thermometer. (8) 
Replace rubber tube and remove last trace of ether by suck- 
ing air through the pipette when it will be dry and ready 
for use. Always dry and clean the pipette after use and 
have it ready for the next time. (When thoroughly dry 
the bead in the bulb of the pipette rolls about freely when 
the pipette is shaken.) 



38 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 



To make the dilution, have the diluting fluid ready. 
Well up a good sized drop of blood (without squeezing 
hard) and draw blood (Fig. 36) into the pipette to the 




Fig. 37. — Filling diluting pipette with diluting fluid. Pipette held high 
enough that the marks maj^ be easily seen. 





Fig. 38. — Revolving pipette to mix contents. Note that pipette is held at about 
the middle. By holding rubber tube as shown, and revolving it by rolling 
the fingers, best agitation and mixing is secured. 

mark 1. Quickly wipe the tip of the pipette to remove 
any blood on it and draw up (Fig. 37) diluting fluid until 
the mixture reaches the mark 101, being careful not to 
get an air bubble in the pipette. Now revolve (Fig. 38) 
the pipette to mix thoroughly and the dilution is ready for 
use. The counting may be done any time within an hour 
or two. If to be carried any considerable distance to the 



BLOOD 39 

laboratory, stretch a rubber band over the ends of the 
pipette (Fig. 39) to prevent the fluid from running out. 







Fig. 39. — Pipette containing diluted blood. Rubber band stretched over ends 
to prevent escape of fluid when carrying. 



Making the preparation. — The counting chamber and 
cover-glass must be scrupulously clean and free from dust 
particles. Since the parts of the counting chamber are 
cemented together it must never be washed with anything 
except water, but it can be washed with water freely. Care- 
fully dry it and then wipe it free of dust particles with a 
handkerchief or similar piece of cloth. If it is held so 
that the light falls upon it at an angle and so that there 
is a dark background in the distance, dust particles can be 
much better seen. These should be picked off with the 
handkerchief wrapped around the end of the finger. Hav- 
ing it clean, rest it face downward against some con- 
venient object, as the foot of the microscope (Fig. 40), 
to prevent dust particles from settling on it before use. 

Now thoroughly clean the cover-glass. Wash it if neces- 
sary. Hold by the edges only (Fig. 40) when cleaning. 
After it has been wiped as clean and free of dust particles 
as possible, with the handkerchief, hold it in proper indirect 
light with a dark background in the distance to see any 
remaining dust particles which should then be picked off. 
Now place it, clean side down, upon some convenient dust 
free surface, like the wheel of the mechanical stage (Fig. 
41), until required for use. 

Having the counting chamber and cover-glass ready 
for use, have convenient some hard glass or metal object 
(preferably not sharp on the edge) with which to press 
down the cover-glass when needed. The handle of the 
platinum loop or eye end of the blood sticker needle or 



40 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 




Fig, 40. — Cleaning cover-glass. Note that it is held by the edges only 




Fig. 41. — Counting chamber resting face downward against the foot of the micro- 
scope after it has been cleaned. Cleaned cover-gl^ss resting upon wheel of 
mechanical stage. 



BLOOD 



41 



even the homo vial which holds it are convenient for this 
purpose. Wood or rubber instruments are not good, nor 
is a lead pencil. 

Now thoroughly mix the contents of the pipette (rubber 
tube attached) and blow out two or three drops of fluid 




Pig. 42. — Squeezing out diluted blood upon the counting plate (old style counting 
chamber), ^fote folding of soft rubber tube. 




Fig. 43. — First step in placing cover-glass upon the counting chamber, 
both hands rest (to steady them) upon the table. 



Note that 



to wash out the long arm of the pipette. Then quickly 
squeeze (Fig. 42) onto the center of the counting plate 
the necessary amount of the diluted blood. A little expe- 
rience is necessary to get the proper amount. It should 
never be sufficient to run over into the moat, but must 
always more than cover the ruled area. Place the cover- 
glass over it at once. The steps in doing this properly are 
illustrated in Figs. 43, 44, 45, 46. The cover-glass should 



42 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 



now be pressed down (Fig. 47) firmly at each of the four 
corners. Do not move the cover-glass in doing this. There 
must never be fluid between the cover -glass and the plates 
on which it rests. The directions sometimes given by manu- 
facturers of the new style counting chambers to allow the 




Fig. 44. — Second step. The cover-glass is tilted up by the pressure of the 
finger upon the edge that extends a little over the outer plate. 




Fig. 45. — Third step. Bringing down the cover-glass by pressure with the 
handle of the platinum loop. 

fluid to flow under the cover-glass is a mistake and should 
not be followed. 

If the surfaces of the plates and cover-glass are 
perfectly clean and free from dust particles, and if the 
manipulation has been properly done, Newton's color rings 
(rainbow colors) will be present on each side. These can 



BLOOD 



43 



c 


•fc 


*^\ 


, ■'">, 




.^^■Hf^^^" ' 




TT^l 







Fig. 46. — Fourth step. 



The cover-glass has been brought down and the finger 
has been lifted from it. 




Fig. 47. — Fifth step. Pressing down the cover-glass at each corner. Note that 
the hand holding the instrument with which pressure is made is steadied by- 
resting upon the table. Care must be taken to avoid slipping the cover-glass. 



44 PKACTICAL CLINICAL LABORATORY DIAGNOSIS 

be seen best by looking at the top of the preparation held 
toward the light of a window or door and just a little 
below the level of the eyes. Unless they are present the 
preparation is not a good one. Do not tilt the 'prepara- 
tion. Hold it level. Now lay it down for two or three 
minutes during which time all the cells settle onto the. 
surface of the counting plate. 

In spite of good technic and usually due to slow manipu- 
lation, the cells sometimes settle unevenly and the next 




Fig. 48. — Looking at distribution of cells upon ruled plate. Dark background 
directly in front and in the distance. Light from window at left. 

step is to examine it to determine this question. Hold the 
preparation (level) above the level of the eyes (Fig. 48), 
having the light fall upon it from above, but look at it 
from below with a dark background in the distance. The 
evenness (Fig. 49) or unevenness (Fig. 50) can be seen, 
and if the cells are not evenly distributed the preparation 
is not good. 

Counting the cells. — Reduce and adjust the light of the 
microscope so that the ruling can be seen best. Count with 
the 4 mm. objective. Find the upper left hand corner of the 
ruled area (in fact the lower right hand corner; images are 



BLOOD 



45 







■-■ ,.:-.'•'" •'. '-'■' }■•' ..:£-" ^<j. -» 









*?$:?;■< -"^ •'■*■• A* "* # ; .. 1 -^^ •••.'••••* --<^ 



Fig. 49. — Even distribution of cells upon the ruled plate. 



















Fig. 50. — Uneven distribution of cells upon the ruled plate. Note thin area at 
top and thick area on the right. 



46 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

reversed in the microscope). Place the end of the first 
rectangle in the center of the field and move toward the 
right, counting all the leucocytes found in the rectangle. 
When the end is reached drop down until the next rectangle 
is in the center of the field and move in the opposite direc- 
tion until all the cells in it are counted. Drop down to the 
next and count all the leucocytes in it in the same way. 
Thus continue until all the leucocytes in the ten rectangles 
have been counted. Make the rule of counting in any cell 
found upon the left or upper boundary line of any square 
or rectangle when counting the cells in that area, but count 
out any cell found upon the right and lower boundary lines 
of a given rectangle or square, when counting the cells in it. 

The leucocytes are stained violet when Toison's fluid is 
used. They are clear, many-shaped, prominently appear- 
ing bodies when acetic acid is used. The small ruling is 
disregarded when counting the leucocytes. 

Multiply the number of leucocytes counted in the entire 
ruled space by 250, which gives the number of cells per 
cu. mm. 

Interpretation of total leucocyte counts. — Blood of nor- 
mal individuals contains 5,000 to 8,000 leucocytes. There is 
a slight increase during digestion, and children's blood gives 
slightly higher values than adults'. 

The total number of leucocytes is increased in leukemia 
sometimes to very high figures. Seven hundred thousand 
has been recorded in myelogenous leukemia, but 400,000 to 
500,000 is an average high count. In lymphatic leukemia 
the count seldom reaches 200,000; 90,000 or 100,000 being 
an average high count. 

The total number of leucocytes is increased in those 
diseases in which there is increase in the percentage of 
particular cells as a result of irritation of the tissue which 
produces them, or as a result of the demands of nature for 
the particular cells to antagonize the disease process pres- 
ent. For instance, there is usually slight increase in syphilis 
during the stage of lymph tissue involvement. In the 



BLOOD 



47 



worst cases of disease due to pyogenic bacteria and in 
which much toxin is absorbed, the total leucocyte count 
may be increased to 40,000, or even 50,000, but these 
figures are rare. Xaturally the count depends not only 

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Fig. 51. — Arrow indicates order in which the erythrocytes in the sixteen small 
squares in one large square should be counted. We count 200 cells here. 

upon the disease or toxin which calls out the cells, but also 
upon the capacity of the individual to meet the demand. 
Long-continued disease often tends to exhaust the capacity 
to respond. 

At the end of this chapter we give a list of the common 



48 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 



diseases and their influences upon the differential and total 
leucocyte count. It should be understood that this gives 
only the general tendency and cannot be exactly correct in 
all cases, and in all combinations of disease. The total 
leucocyte count should be interpreted in the light of the 
other clinical facts in the case as well as the differential 
leucocyte count. 

Counting the erythrocytes, — The same apparatus is used 
as in making total leucocyte counts. Use either Toison's 




oGqo 



o 



Fig. 52. — The twelve cells to be counted in this small square are shaded. 
Those not shaded are not counted. 



fluid or 0.85% salt solution as diluting fluid. Dilute the 
blood one hundred times. When Toison's fluid is used the 
leucocytes and erythrocytes may be counted in the same 
preparation. In either case make the preparation in the 
counting chamber as described for counting leucocytes. 

In the Bass ruling (Fig. 35) there are five large squares, 
— one at each corner of the ruled space and one in the 
center, each ruled into sixteen small squares. Count the 
erythrocytes in each of these large squares. Begin at the 
upper left hand corner and count all the erythrocytes in 
one small square after another (Fig. 51) until all are 
counted. Much time can be saved by counting by twos 
or fives instead of by ones. When considering any given 




PLATE IV. 

TALLQUIST HEMOGLOBIN SCALE SHOWING THE SPECIAL 
BLOTTING PAPER REQUIRED AND THE ARRANGE- 
MENT OF THE COLORED SCALE 

The colors here shown are not exactly the same shade as in the original, 
they do not correspond to the different percentages of hemoglobin and can- 
not be used to ascertain or measure the quantity of hemoglobin. 



BLOOD 49 

square count in (Fig. 51) all cells that touch the left or upper 
lines, and count out all that touch the right and lower lines. 
Do not stop to write down the cells until all in a large 
square have been counted. Add up the number of cells 
counted in the five large squares. The total number of 
cells counted in the five large squares multiplied by 5,000 
equals the number of cells per cubic millimeter. 

Estimation of the hemoglobin, — The Tallquist scale con- 
sists of a series of ten red colored cards (Plate IV), each 
representing different percentages of hemoglobin in steps 
of ten and ranging from 10% to 100%. The colored cards 
are mounted on one sheet and bound in book form, 
with several pages of soft white filter paper. Each colored 
card is perforated in the middle with a hole about one- 
fourth inch in diameter. Well up a good drop of blood 
without much squeezing and touch a piece of the white 
paper to it. A spot on the paper is thus saturated with 
blood. Wait just a moment to allow the blood to spread 
through the paper by capillarity as far as it will and then 
promptly compare it with the different shades of the color 
sheet, holding the paper behind the color and viewing the 
blood through the holes in the color card. Pass quickly 
from one to the other until the blood is matched with a 
shade on the color sheet and note what per cent, it repre- 
sents. This is the estimate of percentage of hemoglobin in 
the blood. Accuracy improves with practice. Sometimes 
the color seems to fall between two cards or shades, and in 
such case the estimate should be made accordingly. For 
instance, if it seems to fall between 80% and 90% we would 
call it 85%. 

Color index. — The color index of blood is an expression 
of the average amount of hemoglobin per erythrocyte, the 
normal being represented by 1. Divide the percentage of 
hemoglobin by the percentage of erythrocytes. The quotient 
is the color index. A quick way to find the per cent, of 
erythrocytes is to multiply the first two figures on the left 
hand side of the number of erythrocytes per cu. mm. by 2, 



50 PKACTICAL CLINICAL LABORATORY DIAGNOSIS 

unless it is less than one million, when only the first figure 
would be multiplied by 2. 

Interpretation of number of erythrocytes, hemoglobin 
per cent, and color index. — In health, females have about 
4,500,000 to 5,000,000 erythrocytes per cu. mm., and males 
have about 5,000,000 to 5,500,000. Five million is generally 
considered the average. 

The hemoglobin in blood of normal individuals is about 
100%, but there is a slight variation above, or especially be- 
low, in most people. Reading with the Tallquist scale we 
may consider values of 80% to 110% within the bounds of 
technical variation and the variation of normal blood. 

The color index of the blood of normal individuals is 
theoretically 1, but there is a slight variation above or below 
this in apparently healthy individuals. Indices of from 0.8 
to 1.15 are within the limits of the variation in the blood of 
normal individuals and of technical variation. 

The number of erythrocytes may be increased by any 
disease or influence that removes considerable water from 
the blood, provided, of course, a corresponding amount is 
not taken in at the same time. Acute diarrhea, profuse 
sweating, etc., may lead to increase in the total number of 
cells per cu. mm. Abstinence from fluids leads to the same 
condition. Profuse diuresis, such as occurs in advanced 
diabetes, usually leads to concentration of the blood cells. 
Decompensation in various heart conditions tends to con- 
centrate the blood and thereby increase the erythrocyte 
count. In addition to these various dehydrating influences 
or diseases which may lead to an increased number of erythro- 
cytes, there is a disease, idiopathic polycythemia, in which 
the total erythrocyte count sometimes reaches as high as 
from 7,000,000 to even 10,000,000 per cu. mm. 

The hemoglobin percentage is increased by any of the 
above influences that increase the erythrocyte count. The 
color index would therefore be unchanged. 

The number of erythrocytes is decreased in all anemias. 
The decrease may be much or little according to the grade 



BLOOD 51 

and kind of anemia. Counts as low as 500,000 sometimes, 
but very rarely, are met. The kind of anemia is indicated 
and the diagnosis much aided by a consideration of both 
the erythrocyte count and the hemoglobin percentage — the 
color index. 

The number of erythrocytes is reduced in the primary 
anemias as, for instance, pernicious anemia, to sometimes 
1,000,000 cells or less; and though the hemoglobin is also 
always below normal, the reduction of the hemoglobin is 
not so great as that of the erythrocytes. This gives rise to 
a high color index (1.15 to 1.65 or more). A color index 
well above the limits of technical and normal variation 
strongly indicates pernicious anemia. 

The number of erythrocytes is somewhat reduced in 
chlorosis, but usually the reduction is only moderate. In 
fact, a great many cases of chlorosis have more than 4,000,- 
000 erythrocytes. The hemoglobin is greatly reduced in 
this disease and this gives rise to a very low color index. 
Such indices as 0.35 to 0.5 are common, and strongly indi- 
cate chlorosis. 

In secondary anemia, due to loss of blood or to disease, 
there is more or less reduction in the number of erythrocytes 
according to what may be the cause in the particular case. 
The hemoglobin is reduced more than the number of 
erythrocytes, giving rise to a low color index. The color 
index is nearly always below the normal limits in secondary 
anemia. In long standing secondary anemia with low hemo- 
globin and erythrocytes the color index may approach or 
reach normal figures. The lowest color index occurs in 
chlorosis. 

Pathological erythrocytes. — The shape of normal eryth- 
rocytes is that of a biconcave disc. Sometimes there is 
great variation in the shape of many of the cells. This con- 
dition is known as poikilocytosis and such abnormally shaped 
erythrocytes are poikilocytes. (Plate V.) 

Sometimes there is great variation in the size of the eryth- 
rocytes. Some are much smaller than the normal and are 



52 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

called microcytes. Others are much larger than the normal 
size and are called megalocytes. This condition of great 
variation in the size of the erythrocytes is called anisocytosis. 
(Plate V.) 

Normal erythrocytes in the circulating blood never have 
nuclei. In certain blood diseases, especially the primary 
anemias, a few to very many nucleated erythrocytes (Plate 
V) are present. The nuclei vary in size. They are round 
or oval, sometimes presenting two or more lobes and in a 
good many of the larger ones a reticulated structure can 
be made out. This gives some suggestion of the spokes of 
a wheel. Nuclei of erythrocytes stain dark blue to almost 
black with Wright's stain and usually appear to be separate 
from or lying upon the cell to which they belong. 

When very large erythrocytes (megalocytes) contain 
nuclei they are called megaloblasts. 

A normal size erythrocyte containing a nucleus is a 
normoblast. 

We sometimes find free nuclei. 

In anemia, due to certain poisons, notably chronic lead 
poisoning, pernicious anemia, the leukemias, and to a less 
extent malaria, a few to many of the erythrocytes contain 
basophilic (blue staining) granules. These vary much in 
size and number from a few coarse granules to many very 
fine granules. Such cells are known as stippled, or granu- 
lar erythrocytes. (Plate V.) 

In normal blood all the erythrocytes take about the same 
shade of color when stained with Wright's (or other poly- 
chrome) stain, but in pathological blood, certain cells are 
often basophilic and take more or less of the blue. These 
cells are known as basophilic erythrocytes and the condi- 
tion of the blood as polychromatophilia. (Plate V.) 

It should be clearly understood that there may be many 
different combinations or variations from the normal in a 
single cell or in the cells in a given blood. For instance, a 
megalocyte may be nucleated and therefore be a megalo- 
blast, it may be a poikilocyte, it may be stippled and it may 



Plate V. 



Anisocytosis. 




m 



Poikilocytes 






P# • 




* 



i 



Megaloblasts. 

* % + • 



% 



; 







# 



Granular or Stippled Erythrocytes 




|| 



'i 



Polv 'vbilia. 






irocytes. 



BLOOD 53 

be basophilic. In fact, many of the nucleated erythrocytes 
are basophilic or stippled or both. 

Interpretation of pathological erythrocytes. — Nucleated 
erythrocytes, poikilocytosis and anisocytosis occur chiefly in 
the primary anemias (pernicious anemia, leukemia, etc.). 
In considering poikilocytosis and anisocytosis due allowance 
must be made for the variation in size and shape that oc- 
curs, especially at the edge of slide preparations of normal 
blood. In the pathological condition the abnormality exists 
in all parts of the preparation. 

Stippled erythrocytes suggest lead poisoning, or pri- 
mary anemia (and rarely malaria). 

Polychromatophilia suggests primary anemia (and 
chronic malaria). 

In many cases pathological erythrocytes appear in 
showers lasting for a few days, and then almost disappear 
for an equal or a longer period. 

The absence of pathological erythrocytes in a case of 
anemia is evidence (not conclusive) of secondary anemia. 
Secondary anemias of long duration occasionally tend to 
approach the blood picture of pernicious anemia. 



CHAPTER III 

MALARIA 

Obtaining blood and making preparations for examina- 
tion for malaria plasmodia. — There are more malaria plas- 
modia in the peripheral blood during the first six to eight 
hours following the onset of each paroxysm than at any 
other time, but there are sufficient plasmodia at all times 
for a diagnosis to be made in practically all cases of malaria 
having fever at the time. The time to obtain specimens of 
blood for examination is therefore whenever malaria is sus- 
pected. Do not wait until any special time with reference 
to the occurrence of paroxysms. 

Make an ordinary blood spread (see page 22). The 
specimen may be stained and examined at once or at your 
convenience, according to the demands of the case. Stain 
with Wright's stain. 

Making the examination and recognition of plasmodia. — 
Search for plasmodia only in parts of the preparation where 
the staining is good (nuclei of the mononuclear leucocytes 
are purple and the cytoplasm definite blue) and where the 
cells are not too thick. Prior to the acquisition of much ex- 
perience with malaria plasmodia never examine where the 
cells are more than one cell thick. After sufficient experience 
you can recognize plasmodia satisfactorily where the spread 
is two or three cells thick and of course at a considerable 
saving of time, since the thicker the blood film the more 
plasmodia are present in a given area. 

Malaria plasmodia are in (or on) the erythrocytes, and 
no object should be considered as probably a plasmodium 
unless it is so situated. It is true that many plasmodia, 
especially old gametes, are in (or on) little more than 

54 



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Quartan Malaria Plasmodia. 



MALAKIA 61 

"shadows" of erythrocytes, the cells having faded to a great 

extent. 

The cytoplasm stains blue, the chromatin stains the 

usual red, like chromatin in the nuclei of cells, platelets, etc. 

Pigment granules when present do not stain but have a 

light to dark brown color. Do not expect to find malaria 

plasmodia well stained in preparations where the leucocytes 

and platelets are not well stained. Unless the nuclei 

of leucocytes have the proper reddish tint due to proper 

staining of the chromatin in them, the chromatin of the 

plasmodia will not be properly and characteristically stained. 

In examining a blood specimen for malaria plasmodia one 

should acquire the habit of noting, upon first look at the 

preparation, whether the staining is good and not look 

for plasmodia in any but well stained preparations and good 

areas of such preparations. 

The number of plasmodia present varies very much. 

Often there may be several plasmodia in every field, while 

in other cases there may be so few that a search of several 

minutes may be required before a single plasmodium is 

found. 

Description of malaria plasmodia. — There are three 
distinct species of malaria plasmodia now known to infect 
man, and it is highly probable that it may be found that 
still others exist. They are (1) Estivo-autumnal (Plate VI, 
Plasmodium falciparum) ; (2) tertian (Plate VII, Plas- 
modium vivaoc) and (3) quartan (Plate VIII, Plasmo- 
dium malaria?). They all grow and reproduce in the 
blood of man in the same general way. The smallest 
Plasmodium consists of a thin round wall of basophilic 
protoplasm enclosing a round or oval mass that does not 
stain. This gives the appearance of a ring. There is 
one, and sometimes there are two granules of red-stained 
chromatin at one side of the ring, giving rise to the designa- 
tion "signet ring." As the plasmodium grows the erythro- 
cyte is slowly consumed and after about twelve to twenty- 



62 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

four hours there appear light to dark brown granules of 
pigment. These become more numerous until the develop- 
ment of the parasite is complete. An individual Plasmo- 
dium reaches maturity in about forty-eight hours in the 
case of estivo-autumnal and tertian, and in seventy-two 
hours in the case of the quartan parasite. The chromatin 
granule grows in mass for about half the developmental 
period, and after this begins to divide. Division goes on 
slowly until at the end of the period it has divided into the 
number of divisions peculiar to the particular species. To- 
ward the end of the period division of the cytoplasm of the 
organism occurs, and now the whole plasmodium is made 
up of a number of separate chromatin granules each sur- 
rounded by its own cytoplasm. Such a mature, or seg- 
mented plasmodium is called a "rosette." Finally the 
capsule of the rosette ruptures and each segment or young 
plasmodium, known as a merozoite, is capable under favor- 
able conditions of attaching itself to an erythrocyte and 
passing through a similar cycle of development, giving rise 
in turn to another crop of merozoites. This kind of re- 
production takes place without sexual influence and is 
known as schizogony. A-sexual plasmodia (of any age) 
are schizonts. 

After a person has been infected with malaria plasmodia 
more than two or three weeks, and as a result of in- 
fluences not now well understood, certain plasmodia, in- 
stead of developing through the a-sexual cycle of schizo- 
gony, become sexually differentiated and take somewhat 
different form. These are supposed not to be capable of 
reproduction in man. The males are capable of giving 
rise (in the mosquito) to microg'ametes, and the females 
to macrogametes. They are capable of reproducing by 
sporogony in the mosquito. Sexual plasmodia in the blood 
are correctly called gametocytes, but in common usage the 
term gamete is used. Estivo-autumnal gametocytes are 
crescent shaped, but tertian and quartan gametocytes are 
round or oval and resemble very much the developing, al- 



MALARIA 63 

most mature schizonts. There is no division of the chro- 
matin in gametes. 

Estivo-autumnal schizonts disappear from the peripheral 
circulation and lodge in the capillaries when they are about 
twelve hours old. They do not reappear in the peripheral 
blood until after segmentation (except extremely rarely) 
and therefore the only estivo-autumnal schizonts found in 
peripheral blood are the ring forms. 

The tertian and quartan schizonts begin to disappear 
from the peripheral circulation after about two-thirds of 
their period of development has passed, but on account of 
the fact that they are capable of ameboid movement they 
often pass out of the capillaries in which they lodge and 
reappear in the peripheral circulation. It frequently occurs 
that full grown schizonts and rosettes of these species are 
found in the peripheral circulation. This fact that all ages 
of tertian and quartan schizonts are present in peripheral 
blood and only the ring stage of estivo-autumnal schizonts 
constitutes a most valuable means of differentiation between 
the different species. 

The gametes of all species appear in the peripheral 
blood. 

Differentiation of malaria plasmodia. — The rings of dif- 
ferent species look very much alike and it is not practical 
to diagnose species by the rings. The very small rings 
found in pernicious estivo-autumnal malaria may be dif- 
ferentiated from those of the so-called benign estivo- 
autumnal or tertian and quartan parasites. They are 
extremely small and are often overlooked. The presence of 
other size parasites than rings shows either tertian or 
quartan infection. The absence of them indicates that the 
rings are estivo-autumnal. 

Infection with more than one species occurs, but is rare. 
The most useful differential points are shown in the fol- 
lowing table: 



64 



PKACTICAL CLINICAL LABORATORY DIAGNOSIS 





Estivo- Autumnal 


Tertian 


Quartan 


Schizonts in the pe- 


Rings only. 


All sizes. 


All sizes. 


ripheral blood. 








Shape of gametes. 


Crescentic and 
oval. 


Round or oval. 


Round or oval. 


Shape of outline of 


Rings. May have 


Irregular after 


Regular after ring 


schizonts in periph- 


two chromatin 


ring stage 


shape is passed. 


eral blood. 


granules. 


passed. 




Influence upon color of 


Not changed. 


Faded, pale. 


Darker. Normal 


erythrocyte. 






red color inten- 
sified. 


Influence upon size of 


Not changed. 


Enlarged. 


Reduced. 


erythrocyte. 








Pigment in schizonts 


Medium to coarse. 


Very fine. 


Coarse. 


and gametocytes. 








Schiiffner's granules.* 


(Not present.) 


Often present. 


Not present. 


Number of segments in 


(24 to 32.) 


32. 


8. 


rosette. 








Period of a-sexual de- 


(48 hours. ) 


48 hours. 


72 hours. 


velopment. 









* Red staining granules in the erythrocyte containing the parasite. 

Interpretation of examination of the blood for malaria, 
— There are always sufficient plasmodia in the peripheral 
blood to enable one who is competent to find them in a 
search of ten minutes or less in the case of all persons who 
have active malarial fever. It is possible, therefore, to de- 
termine positively that a given case of fever is (or is not) 
due to malaria. If quinine has been taken by the patient 
during the 48 hours previous to the time the blood is taken 
tor examination it may cause the disappearance of plas- 
modia, and the question as to whether quinine has been taken 
recently should always be raised in interpreting negative 
findings. 

It is often possible to find plasmodia in the blood of 
people who have chronic or latent malaria by thorough and 
repeated examinations of the blood, but it is not possible 
to examine the blood and to determine that the patient has 
not some plasmodia in his body. If he had enough to make 
him sick, however, they could be found readily. It should 
be appreciated that the number of plasmodia present in the 
blood is not always an indication of the clinical symptoms 
or vice versa. 



CHAPTER IV 

TYPHOID AGGLUTINATION TEST 

This is a test to determine the presence or absence in the 
blood of a specific substance, agglutinin, which has the 
property of causing typhoid bacilli to agglutinate or collect 
together in clumps. Typhoid agglutinin will agglutinate 
either living or dead typhoid bacilli. The method of making 
the test given below is as reliable as any other when done 
with corresponding accuracy and reliable material, and it 
has many advantages. 

The special material required is a suspension of dead 
typhoid bacilli, ten billion per c.c. in distilled water, and 
killed and preserved with 1% commercial formalin. A 24 
hour growth of typhoid bacilli on neutral agar is used in 
making it. This material can be made by any competent 
bacteriologist. This material keeps well for at least several 
years if kept tightly corked. Shake before using. When 
a vial of it is frequently opened and used from, it should be 
discarded and a new supply obtained every six to twelve 
months. An ordinary medicine dropper and a wooden tooth- 
pick are also needed. 

Make the regular blood-spread on a slide, using approxi- 
mately one-fourth drop of blood. This may be tested at 
once at the bedside, or it may be tested at any time at your 
convenience, within a week or two. Place on the blood one 
drop of water. Carefully spread this over the film of blood 

65 



66 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 



(Fig. 53) with the end of a pick or other suitable instru- 
ment. Tilt the slide from side to side to hasten the dis- 
solving of the blood. A clear solution of approximately 








Fig. 53. — Spreading the drop of water over the blood to dissolve it in 
making typhoid agglutination test. 

one-fourth drop of blood in one drop of water is thus made. 
Now add one drop of the suspension of typhoid bacilli and 
mix by tilting the slide from side to side (Fig. 54) and 
from end to end, causing the mixture to flow back and 




Fig. 54. — Tilting slide back and forth to facilitate mixing and to hasten 

agglutination. 

forth. This also hastens the reaction. If the blood con- 
tains the specific agglutinin for typhoid bacilli, the millions 
of bacilli present soon begin to collect together, first in small 



TYPHOID AGGLUTINATION TEST 



67 



grayish clumps (Fig. 55) appearing as a fine granular sedi- 
ment. Later the clumps of bacilli get coarser and are 
readily recognized (Figs. 56 and 57). The reaction takes 
place and is complete within two minutes. When the test 
is negative no such granular sediment forms. 

Practice with known typhoid and known negative blood 
until familiar with the test. Make many such tests until 
familiar with the appearance of positive and negative re- 
actions. Do not mistake dust particles for agglutinated 




Fig. 55. — A series of typhoid agglutination tests, (a) Negative test, (b) Weak 
positive reaction, (c) Moderate positive reaction. (d) Strong positive 
reaction. 

bacilli. There occur doubtful reactions in this test just 
as in all other tests for specific antibodies which vary in 
amount from none to sufficient to produce definite positive 
reactions. 



Interpretation of the typhoid agglutination test. — About 
10 to 20% of all cases of typhoid give positive reactions by 
the end of the first week. About 70% give positive reactions 
by the end of the second week. More than 90% give posi- 
tive reactions at some time during the course of the disease. 
A few do not give positive reactions at any time. Usually 



68 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 



the reaction may be obtained until after the fever sub- 
sides. The reaction gradually gets weaker as time passes 
following convalescence, and after three to six months the 




Fig. 56. — Looking at typhoid agglutination test. The granules can be seen best 
when there is a dark background in the distance and the light falls upon 
the specimen from the side. 

reaction is weak or negative in the majority of cases. A 
few give positive reactions for a year or two. 

A positive reaction, if accompanied by clinical evidence, 
is practically diagnostic of typhoid. A negative reaction 
does not prove that the patient has not typhoid. Its nega- 
tive value is in proportion to the duration of the disease. 
Given a case of fever (possibly typhoid) of two weeks' 
duration, for instance, the indication of a negative reaction 



TYPHOID AGGLUTIXATIOX TEST 



69 




Fig. 57. — Looking at typhoid agglutination test at night by the light of a match 
held in the right hand beneath and to the side of the specimen. 



would be 70% that it is not typhoid. Here, as in most 
other laboratory findings, the test is of most value when 
interpreted in connection with the clinical evidence. The 
same test and technic may be used in the diagnosis of 
paratyphoid fever provided one uses similar suspensions 
of paratyphoid bacillus A, and paratyphoid bacillus B, in 
place of the suspension of typhoid bacilli. 



CHAPTER V 

URINE 

Collection of specimens for examination. — The only 
proper specimen of urine for examination is a recently 
voided specimen, except perhaps when it may be desirable 
to make a quantitative examination of the total urine voided 
during twenty-four hours. No method of preservation will 
keep specimens in suitable condition for examination. Dif- 
ferent specimens keep variable lengths of time. Some are 
not good after an hour or two, while others are satisfactory 
for at least ten or twelve hours, and sometimes longer. As 
specimens get a few hours old, contaminating bacteria, which 
often multiply very rapidly, alter the formed elements, such 
as blood cells, casts, etc., that may be present, cause the crys- 
tallization and precipitation of the salts and effect, by fermen- 
tation or otherwise, such substances as sugar and albumin 
that may be present. Sometimes the amorphous or crystal- 
line sediment that forms in a short time is so great that it 
renders microscopic examination impractical or at least unre- 
liable. Do not examine old specimens unless as a last resort. 

Patients who come to the laboratory or office should 
void urine there for examination, but if inconvenient they 
may be instructed to void just before leaving to come to the 
office and to bring a sample of the fresh urine only. Speci- 
mens to be sent to the laboratory should be voided just be- 
fore they are sent. Examination of the early morning urine 
is sometimes the most valuable. Instruct the patient to 
void in a clean vessel or urinal and to send about four ounces 
in a clean bottle. Larger quantities are unnecessary. 

If significance is to be attached to the presence of pus, 
instruct the patient to void if possible into two clean glasses 
and to send two to four ounces from the second glass in a 
clean bottle for examination. Females should be instructed 
further to thoroughly wash the vulva with soap and water 
before voiding. Some physicians take precautions in all 
cases as routine. However, catheterized specimens are the 
only kind from females that can be depended on as certain 
not to contain pus and acid-fast bacilli from the vulva. 

70 





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PLATE IX 
CHEMICAL TESTS OF URINE 

Tube 1. Test for albumin. No cloud after boiling and addition of acetit 
acid. 

'lube 2. Test for albumin. Cloud produced by boiling and remaining 
after addition of acetic acid, thus showing presence of albumin. 

Tube 3. Test for indican. Negative reaction. Chloroform not colored. 

Tube 4. Test for indican. Positive reaction. Chloroform colored blue. 

Tube 5. Test for sugar with Folding's solution. Negative reaction. 
No change in color. 

Tube G. Test for sugar with Fehling's solution. Positive reaction. 
Yellowish red color due to reduced copper. 

Tube 7. Test for acetone. Negative reaction. Disappearance of red 
color upon addition of acetic acid. 

Tube 8. Test for acetone. Positive reaction. No disappearance of red 
color upon the addition of acetic acid. 



URINE 

Physical Examination 



71 



Specific gravity. — A urinometer (Fig. 58) is required. 
One of the cheaper grades is sufficiently accurate for ordi- 
nary purposes. Fill the tube nearly full of urine so that 
it runs over (in the sink) when the 
float is put in. This makes the read- 
ing easier. Read at the bottom of the 
meniscus. 



Chemical Examination 



Test of the reaction. — Dip a strip 
of blue and a strip of red litmus paper 
in the urine. If acid, the blue paper 
changes to red. If alkaline, the red 
paper changes to blue. 

Test for albumin. — (A) Qualita- 
tive. Fill a test-tube three-fourths full 
of urine. Hold by the lower end in 
the hand. Boil (Fig. 59), for a minute 
or two, approximately the upper one 
inch of the column by holding in the 
flame of a small Bunsen burner or al- 
cohol lamp. Revolve and agitate while boiling to prevent 
boiling over. If albumin is present, a cloud (Plate IX), 



A 



Fig. 58 



Urinometer. 
Testing specific grav- 
ity. Note curve of 
meniscus. 




Fig. 59. — Boiling urine in test for albumin. 



72 



PKACTICAL CLINICAL LABORATORY DIAGNOSIS 



varying in intensity with the amount present, will appear. 
Phosphates also produce a cloud upon boiling. Now add 
a few (4 or 5) drops of glacial acetic acid (Fig. 60). If 
the cloud is due to albumin it will be intensified. If it is 
due to phosphates it will be cleared up. A faint cloud 
due to only a trace of albumin can best be seen by holding, 




Fig. 60. — Adding acetic acid to urine from a drop bottle in testing for albumin. 



the tube (Fig. 61) so that it has a dark background a few 
feet away and has light (from window or otherwise) fall- 
ing upon it from the side (Fig. 62). 

(B) Quantitative. There is no practical quantitative 
test for albumin that is absolutely accurate. The one here 
given is accurate enough for practical purposes. Fill a 
test-tube with urine to a depth of 2% inches (Fig. 65). 
Add about one-fourth as much of a 10% solution of potas- 
sium ferrocvanide and about 1 c.c. of glacial acetic acid. 



URINE 



73 




Fig. 61. — Looking for faint cloud in test for albumin. Dark background 
with light falling on the tube from the side. 




Fig. C2. — Cloud due to albumin in urine as seen in the proper light 
against a dark background. 



74 



PKACTICAL CLINICAL LABOKATORY DIAGNOSIS 



Shake and allow to stand a few (2 to 5) minutes. Cen- 
trifuge until the coagulated albumin has collected well in 
the bottom of the tube. Measure this. Each one-quarter 
inch of sediment represents 10 % of moist albumin. Smaller 
quantities may be approximated. The formula for the po- 
tassium ferrocyanide solution is: 

5 Potassium ferrocyanide 10 gms. 

Water, to make 100 c.c. 

Mix. 




Fig. 63. — Filling tube with urine to a depth of 2% inches in quantitative test for 
albumin. The tube is held by the side of an ordinary inch rule. 

Test for sugar. — (A) Qualitative. There are several 
tests to select from. We give Fehling's test here because 
it is the one generally required by life insurance companies, 
for whom no doubt many of our readers will be called upon 
to make examinations. We require two separate stock so- 
lutions, Fehling's alkaline solution and Fehling's copper 
sulphate solution. The formula for Fehling's alkaline solu- 
tion is : 



URINE 75 

Sodium-potassium tartrate 173 grams 

Sodium hydroxide 125 grams 

Water 500 c.c. 

Mix and filter. 

The formula for Fehling's copper sulphate solution is: 

Pure crystals of cupric sulphate. . . . 36.4 grams 

Water 500 c.c. 

Mix and filter. 

These two separate solutions keep well but when mixed 
together they do not keep more than a few days. It is 
therefore desirable to prepare the test solution at the time 
a test is made by mixing equal parts of the alkaline solu- 



* v 




Fig. 64. — Tube contains Fehling's copper solution. Adding equal amount of 
alkaline solution, using finger as a marker. 



tion and the copper solution. Keep the separate solutions 
in glass stoppered drop-bottles. The drop-bottles are more 
convenient to pour the solutions from. 

To make a test, drop into a test-tube a few drops, not 
more than one-half c.c, of one solution, hold the finger as a 
marker (Fig. 64) at the place where the solution will rise 
to when approximately an equal volume of the other solu- 




76 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

tion is added. Add the 
other solution, shake, and 
we now have about 1 c.c. of 
a mixture of approximately 
equal parts of the two dif- 
ferent solutions, which con- 
stitutes Fehling's test solu- 
tion. Heat the solution to 
boiling over a Bunsen 
burner (Fig. 65) or alcohol 
lamp, then add about one- 
half as much urine and boil 
again. If sugar is present 
the solution assumes an 

Fig. 65.-Boiling Fehling's solution opaqile yellow Color (Plate 

and urine in testing for sugar. r ^ J v 

IX) and shortly after a 
dense yellowish-red sediment falls to the bottom. Shake 
the tube while heating to prevent boiling or "popping" 
out. No tube-holder is necessary. 

(B) Quantitative test for sugar. Measure one-half 
c.c. of each of the Fehling stock solutions into a test 
tube by means of a one c.c. graduated pipette. Wash the 
pipette which should be graduated in hundredths by running 
water through it. Take up some of the urine to be tested 
in the pipette and after noting the starting point add a few 
hundredths of a c.c. to the solution at a time (Fig. 66), 
boiling a little between each addition. Touch the tip of 
the pipette to the side of the tube so that all urine let out 
will flow into the tube. The yellowish red precipitate forms 
and finally the solution loses its blue color at the point 
where all the copper is oxidized. Complete reduction of 
the copper is best determined by centrifuging the tube to 
throw down the precipitate and noting when the blue color 
disappears. Now read off on the pipette the amount of 
urine used and calculate the per cent, of sugar indicated 
by the test. One c.c. Fehling's solution requires 0.005 
gram of glucose to remove the blue color, and therefore 



URINE 77 

the amount of urine used contains 0.005 gram of glucose. 
To calculate the per cent, of glucose present by a short rule, 
divide 0.5 by the amount expressed in c.c. of urine used. 
The quotient is the per cent, of glucose present. For ex- 
ample, if 0.08 c.c. of urine is required, 0.5 -r- 0.08 = 6^, 
the per cent, of sugar indicated. If a large amount of 
glucose is present it is better to dilute the urine, say ten 

r 




Fig. 66. — Adding urine from 1 c.c. graduated pipette in quantitative test 

for sugar. 

times with water and to test this diluted urine. Of course 
the amount of sugar present in the diluted urine must be 
multiplied by the number of times the urine was diluted, 
to determine the amount in the undiluted urine. 

Test for indican. — Place about 3 c.c. of urine in a test- 
tube. Add an equal quantity of hydrochloric acid and about 
1 c.c. Fehling's copper solution. Shake vigorously for one- 
half minute and add a few drops of chloroform. Shake well 



78 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

again and allow the chloroform to settle to the bottom. If 
indican is present the chloroform assumes a blue color ( Plate 
IX), varying in intensity with the amount present, but it 
remains uncolored if indican is not present. A red color 
indicates the presence of iodides. 

Test for acetone. — To about 5 c.c. of urine in a test-tube 
add an amount of sodium nitro-prusside about as large as 
the head of a match, shake to hasten solution and add one- 
half to one c.c. of Fehling's solution (Alkaline). The mix- 
ture at once turns a cherry red (Plate IX), which rapidly 
changes to yellow in the absence of acetone while the color 
changes much more slowly (3 to 5 minutes or longer) if 
acetone is present. Immediately after the alkaline solution 
has been added and the tube shaken and before the red color 
has had time to fade, add a few drops (2 or 3), of glacial 
acetic acid. The color promptly disappears in the absence 
of acetone, but is changed slightly to a darker or purplish 
red when acetone is present. 

Microscopic Examination 

Preparation of specimens.- — The objects which we ex- 
pect to find by microscopical examination are suspended in 
the urine and must be concentrated by means of a centri- 
fuge. Satisfactory examination is impossible otherwise. 
The electric centrifuge is the best ( Fig. 67 ) . It should 
have a guard (Fig. 68) around it to protect against acci- 
dents. A good hand centrifuge (Fig. 69) does well in the 
hands of those who take proper care of them and run them 
right. They do not last long if run with tubes of unequal 
weight in them or if turned by jerks at the very highest 
speed possible. Neither is necessary. The centrifuge should 
be supplied with square bottom Cornell shields with rubber 
washers in the bottom. Do not accept any other. No spe- 
cial centrifuge tubes are required. Use the regular 12 mm. 
X 115 mm. lipless tubes. However, with low speed and 
poor centrifuges the special narrow bottom tubes and cor- 
responding shields are better. Agitate the urine to stir up 



URINE 



79 




Fig. 67. — Electric centrifuge with Cornell shield.' 




Fig. 68. — Box guard around electric centrifuge. 



80 PEACTICAL CLINICAL LABORATORY DIAGNOSIS 

any sediment that may have settled to the bottom of the 
container and fill the tube about two-thirds full. Select an- 
other tube as nearly the same size as possible and fill it with 
water to exactly the same level (Fig. 70), holding them 
side by side for comparison. If two specimens are to be 
centrifuged each tube may be filled with urine and (one) 
labelled. Centrifuge at full speed one-half to two minutes. 



. i 



Fig. 69. — Hand centrifuge with Cornell shields. 

Longer is unnecessary, except when endeavoring to throw 
down bacteria. This cannot be done satisfactorily with the 
hand centrifuge and requires several minutes with the elec- 
tric centrifuge. If not sufficient sediment is obtained from 
the first tube the supernatant fluid may be poured off, leav- 
ing the sediment in the bottom, the tube refilled with urine, 
and centrifuged again. In this way all the suspended ob- 
jects in several tubes of urine may be obtained if desired, 
but generally one tube is sufficient for all practical pur- 
poses. 



URINE 



81 




Fig. 70. — Filling balance tube to height of column of urine. 

After centrifuging, pour off the supernatant fluid. The 
sediment and a drop or two of urine remain. Shake the 
tube so as to break and stir up the sediment and pour it 
out on a slide. Spread out properly on the slide with the 
edge of the mouth of the tube. (Fig. 71.) A proper 




Fig. 71. — Pouring out sediment and spreading on slide with mouth of the tube. 
No pipette is necessary as is also the case with the sediment from feces. 



82 PKACTICAL CLINICAL LABORATORY DIAGNOSIS 

spread (Fig. 72) will not extend to the edges or end of 
the slide. It is the heighth of laboratory awkwardness to 
allow urine to run over onto the microscope stage, which will 




Fig. 72. — Proper spread of urine sediment. 

occur if it is spread to the edge or end. No cover-glass is 
necessary. 

Method of examination. — Examine with the low power 
(16 mm.) lens. It will occasionally be necessary to swing in 
place the high dry lens (4 mm.) to examine under higher 
magnification some particular object about which doubt 
exists. Here as well as in other places it is important to 
have a high power lens with a long working distance, such 
as the B. and L., 4 mm. of N. A., 0.65 (and not one of 
0.85) or the Leitz No. 6 (and not No. 7). Those who, 
through mistake or otherwise, have purchased lenses with 
short focal length should exchange them or procure proper 
ones in some way. Do not try to do routine laboratory 
work under such a handicap. 

Place one corner of the preparation in position for ex- 
amination and by means of the mechanical stage move the 
slide from end to end, dropping down the width of a field 
each time until the entire specimen is examined. This re- 
quires only a minute or two where the examiner is competent. 

The light of the microscope must be adjusted (reduced) 
so that hyaline objects and those with little color may be 
seen well (Fig. 10). 

Diagnosis of microscopic objects commonly found in 
urine*. — Red blood cells (Plate X, b) are usually more or 
less altered by the urine. They may be swollen or more 
or less faded, or they may be crenated. They appear green- 
ish instead of red. They may be differentiated from other 
objects when in doubt by mixing a little acetic acid with 




PLATE X 
MICROSCOPICAL FINDINGS TV URINE 

a. Low power fi"l<l of urine sediment, containing a few epithelial cells, 
pus cells, red blood cells and three hyaline casts. 

b. Higher magnification. Field contains pus cells, red blood cells and 
a few bacilli. 

c. Hyaline casts. Note irregularity in size and shape of different casts. 
Two epithelial cells are shown. 

d. Epithelial cell casts, coarse granular casts and blood cell casts. 

e. Fine granular and hyaline casts. 

f. Cylindroids. 



URINE 83 

the material on the slide. Red blood cells are dissolved, 
while other cells can be seen better. 

Pus cells {Plate X, b) are to be recognized by their 
granular protoplasm and their nuclei which can be made 
out upon proper adjustment of the light. When in doubt 
add a drop of acetic acid to bring out the nuclei better or 
the urine may be drained off after the cells settle on the 
slide and the specimen stained. (Technic, page 140.) 

Epithelial cells (Plate X, a) vary much in their size, 
shape and appearance according to what part or parts they 
come from. They are to be recognized by their distinct 
relatively small single nuclei. 

Casts (Plate X, a, c, d, e) are of several varieties. 
They are casts of uriniferous tubules and therefore vary in 
size and shape according to the size and shape of the differ- 
ent tubules in which they are formed. As the casts are 
forced out of the tubules they are broken more or less so 
that some are short pieces while others are very long and 
many others are of intermediate lengths. Some are two 
or three times as thick as others. The basis of all casts is 
a hyaline material. Many of them consist of this material 
only and are called hyaline casts. 

Sometimes the disease involving the epithelial cells lin- 
ing the tubule where a cast forms results in the loss of the 
cement substance holding the cells in place and some of 
them adhere to the cast when it is forced out. These cells 
are usually swollen and colored more or less yellowish or 
brownish. Such casts are called epithelial casts. An epi- 
thelial cast is a hyaline cast onto which epithelial cells are 
attached. 

If such damaged cells are not carried out with casts 
they may continue to degenerate and break up into coarse 
granules. These adhere to casts that form in the tubules 
and such casts are now called coarse granular casts. The 
granules vary from a light yellow to dark brown in color. 
A coarse granular cast is a hyaline cast with coarse granules 
adhering to or imbedded in it. 



84 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

Unless the granules are carried out while they are 
coarse they continue to break up into finer granules and 
casts forming in a tubule where such a process is going on 
would have some fine granules on them. In fact, granules 
are actually imbedded in the casts. Such casts are called 
fine granular casts. A fine granular cast, therefore, is a 
hyaline cast with fine granules on or in it. 

Sometimes the. degeneration of the lining cells of tubules 
which generally gives rise to epithelial casts, coarse granular 
and fine granular casts is a fatty degeneration instead of 
granular degeneration and casts formed in such tubules 
would then have fat droplets on or in them. Such casts 
are called fatty casts. A fatty cast is a hyaline cast with 
fat droplets on or in it. 

It sometimes occurs that red blood cells pass into tubules 
where casts are forming and adhere to them. Such casts 
are called red blood cell casts. Likewise occasionally pus 
cells may be attached to casts where they form. Such casts 
are called pus cell casts. 

There are often various combinations of the above differ- 
ent varieties of casts. For instance, a single cast may have 
epithelial cells, coarse granules, fine granules, and fat drop- 
lets on or in it. It often occurs that one end of a cast may 
be hyaline while the other may be loaded with granules. 

Rarely casts consist of a highly refractile amyloid ma- 
terial instead of the usual hyaline material. Such casts 
are called amyloid casts. 

In addition to the above true casts there are present in 
most fresh specimens of urine a variable number of shreds 
or cylindroids (Plate X, f ) of a hyaline material very much 
resembling hyaline casts. They are irregular in shape and 
size and one end is usually very thin. They are much 
narrower in places than in others and do not give one 
the impression that they are true casts of the uriniferous 
tubules. 

The dividing line between hyaline casts and some of these 
cylindroids is difficult to draw. In fact one must often 



URINE 85 

feel in doubt about some of them. When in doubt give the 
patient the benefit of the doubt. 

Interpretation of urine examinations. — Specific gravity 
is high (above 1025) in most cases of glycosuria and very 
low in various nervous conditions. A low specific gravity 
(1000 to 1010) with inability to concentrate occurs in in- 
terstitial nephritis. Normal values or higher occur in paren- 
chymatous nephritis. The specific gravity is susceptible of 
great variation in health according to the amount of water 
taken in and the amount of solids excreted. 

The reaction of normal urine is acid. Alkaline urine 
results from an almost exclusive carbohydrate diet. De- 
composition of urine, either in the bladder or after it is 
voided, usually changes the reaction to alkaline. 

Albumin in urine is derived from the blood serum and 
when present indicates that on account of some influence 
serum is passing through the kidneys. High blood pres- 
sure often causes a small amount of albumin in the urine. 
The chief cause of albuminuria is disease involving the 
kidneys. Nephritis is accompanied by albuminuria, the 
amount of albumin varying greatly with the different forms 
of nephritis and in different individuals. Albumin in the 
urine is also produced by inflammatory disease accompanied 
by suppuration involving the mucous membrane of any part 
of the urinary tract. The albumin is derived from the in- 
flammatory exudate and naturally varies in amount greatly. 

Sugar (glucose) in the urine results from diseases and 
conditions involving the metabolism of carbohydrates. Ex- 
cessive eating of sugar may produce it temporarily in in- 
dividuals who are apparently healthy. Diseases and injuries 
involving the floor of the fourth ventricle are usually ac- 
companied by glycosuria. The chief cause of glycosuria is 
diabetes mellitus, a disease of the pancreas involving the 
islands of Langhans. 

An excess of indican in the urine indicates absorption 
of products of putrefaction of animal proteids in the ali- 
mentary tract. Intestinal obstruction or stasis is an im- 
portant cause. 



86 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

Acetonuria indicates "acid-intoxication," whatever that is. 

Red blood cells in the urine show that hemorrhage is 
going on somewhere along the urinary tract. The presence 
or absence of erythrocytes in the urine is very valuable in 
differentiating "renal colic" from other pain due to disease 
in other organs in the same region. 

Pus cells in urine indicate suppuration somewhere along 
the genito-urinary tract. There are a few or many pus 
cells from the vagina and vulva in the urine of most women. 

Epithelial cells are present in all specimens of urine. 
Many more are present in urine from females. 

A few hyaline casts are present in the urine of most 
people past the age of fifty, especially if the blood pressure 
is high. A few may also be found in the urine of persons 
who have not any recognizable disease. Hyaline casts there- 
fore do not indicate necessarily any disease of the kidney. 
On the other hand, they are present and usually in large 
numbers in nephritis of all types. 

Epithelial and coarse granular casts indicate acute 
nephritis. Fine granular casts are present in all forms of 
parenchymatous nephritis, but generally speaking they in- 
dicate somewhat less acute disease than the coarse granular 
and epithelial cell casts do. 

Pus cell casts indicate production of pus in the tubules. 

Red blood cell casts indicate that blood cells are leaking 
through the walls of the tubules at the time and place casts 
are being formed. 

Cylindroids have no pathological significance that we 
know of. 




1: 


_ 



PLATE XI 
TEST OF GASTRIC JUICE FOE, FREE HC1 AND TOTAL ACIDITY 

Tube 9. Clear gastric juice. 

Tube 10. Change of color, upon addition of dimethylaminoazobenzol, 
indicating free HC1. 

Tube 11. HC1 has been neutralized by the addition of decinormal 
sodium hydroxide solution. There is usually a yellowish tint, due to organic 
acids. 

Tube 12. Neutralization of all acids (total acidity). Color produced 
by phenolphthalein indicator. 



CHAPTER VI 

GASTRIC CONTENTS 

Obtaining material to be tested. — After the patient has 
fasted twelve hours (over night) or longer, give a test meal, 
consisting of two or three crackers or a slice of bread and 
one or two glasses of water. The food should be thoroughly 
masticated. After three-fourths to one hour remove the 
stomach contents with the stomach-tube. A tube with a bulb 
to make suction with is quite an advantage. 

Test for free HCl and total acidity. — Obtain some clear 
gastric juice by centrifuging a tube of the gastric contents 
just removed. The heavy particles go to the bottom. The 
froth, etc., rises to the top. Remove one c.c. of the clear 
fluid with a graduated pipette (1 c.c.) and place in an- 
other test-tube. Add one drop of 0.2% solution of dimeth- 
ylaminoazobenzol in alcohol. Formula: 

Dimethylaminoazobenzol 0.2 gm. 

Alcohol (95% ) 100 c.c. 

Mix. This solution keeps well. 

If free HCl is present a bright cherry red color (Plate 
XI, 10) will appear at once. Now add one drop of 0.2% 
solution of phenolphthalein in alcohol. Formula: 

Phenolphthalein 0.2 gm. 

Alcohol (95% ) 100 c.c. 

Mix. This solution keeps well. 

No considerable change occurs. It is now ready for 
titration with decinormal alkali solution, the dimethylami- 
noazobenzol serving as an indicator for the free HCl and 
the phenolphthalein for the total acidity. 

87 



88 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

Now take up in a 1 c.c. pipette graduated in hundredths 
1 c.c. of decinormal sodium hydroxide solution. Formula: 

Sodium hydroxide 0.4 gm. 

Water . . . . 100 c.c. 

Mix. 

Add this slowly to the fluid, shaking as each 0.1 c.c. 
or less is added. Note the amount of alkali required to 
just cause the disappearance of the cherry red color (Plate 
XI, 11). This indicates the total free HC1 present. Con- 
tinue to add the alkali solution until a permanent rose color 
(Plate XI, 12) appears. This indicates the total acidity 
and the total amount of alkali used should be noted. The 
degree of acidity is expressed in terms of the number of 
c.c. of the decinormal solution, that would be required to 
neutralize 100 c.c. of gastric fluid. Each one hundredth c.c. 
in our test represents one degree. For instance, if 0.35 c.c. 
of the decinormal solution is required in the first titration 
the total free HC1 would be 35, and if 0.65 c.c. (in all) is 
required before the second reaction is obtained, the total 
acidity would be 65. 

Other examinations. — Test gastric contents for occult 
blood in the same way as feces are tested for it, see page 107. 

Blood and pus cells can be recognized by microscopic 
examination. Look first at a specimen of the material with 
the high dry objective. If in doubt as to the diagnosis of 
pus cells, make a spread on a slide, and stain (technic, 
page 112) and examine with the oil immersion lens. 

Interpretation of the findings in gastric contents. — The 
free HC1 in gastric contents of normal individuals is usu- 
ally between 25 and 50, by this test. Above 50 indicates 
hyperchlorhydria and occurs in neuroses, most cases of 
gastric ulcer and beginning chronic gastritis. Values below 
25, hypochlorhydria, are usually found in early carcinoma 
and in most conditions associated with general systemic 
depression, including some neuroses. Absence of free HC1, 



GASTRIC CONTENTS 89 

achlorhydria, occurs in most cases of advanced gastric cancer 
and far advanced chronic gastritis. 

Total acidity is due to HO, free or combined, to acid 
salts and in pathological conditions to organic acids. Nor- 
mally it ranges from 40 to 70. Variations above or below 
these figures have the same indications as variations in the 
free HO, provided there is a corresponding increase or 
decrease of the free HC1 present. Whenever the free HC1 
is very low or absent and the total acidity is high, it indi- 
cates fermentation with the production of organic acid, 
such as occurs in advanced carcinoma and far advanced 
chronic gastritis. 



CHAPTER VII 

FECES 

Examination for intestinal parasite ova and larva?. — 
All the intestinal worms either lay eggs or deposit larvae 
in the intestinal canal. These may be found in the feces. 
Most of the worms produce very large numbers of ova or 
larvae. They are characteristic of the particular kind of 
worm that produces them. Whether a given individual is 
host for intestinal worms can therefore be determined by 
microscopic examination of the feces for ova and larvae. 
The kind of worms present can also be determined by the 
same means. 

Collection of specimens. — All of the intestinal worms 
commonly found in this country inhabit the small intestine 
and cecum, except oxyuris, which inhabits the large intes- 
tine. The ova and larvae being deposited high up in the 
intestine are thoroughly distributed in the feces. Oxyuris 
ova are sometimes more numerous on the surface of formed 
stools. Feces of normal consistency are most suitable for 
examination. Liquid feces generally contain fewer ova, 
but they usually contain a sufficient number for diagnostic 
purposes. 

One or two drams of feces is a sufficient quantity. 
Larger quantities are objectionable and sometimes un- 
pleasant and embarrassing. Unless patients are instructed 
as to the quantity desired and how to collect it they some- 
times bring to the laboratory very large quantities and in 
the most inappropriate containers. A fruit jar full of 
liquid feces is not at all uncommon; and an unwieldy pack- 
age it is. Or a bottle is completely filled with fermenting 
feces and tightly stoppered. The shaking during transpor- 
tation hastens gas formation, and whenever the stopper is 
loosened it often shoots out, scattering feces about the labo- 
ratory and sometimes on the operator. A wide mouth two 

90 



FECES 



91 



ounce bottle (Fig. 73) with a new cork stopper is a very 
appropriate container. The patient may be instructed to 
obtain such a bottle from the drug 
store, or it should be furnished 
him at the time the specimen is 
requested. At the time the con- 
tainer is supplied and the speci- 
men requested it is well to tact- 
fully tell the patient that only a 
small quantity — "about so much" 
— is desired, and not to fill the 
bottle full. Specimens should be 
submitted for examination within 
twenty-four hours of the time they 
were passed whenever practical, to 
facilitate the recognition and dif- 
ferentiation of strongyloides larvae, 
but much older specimens are satisfactory to examine 
for ova. 

Making the preparation for examination. — Place two 
drops (not more) of water in the middle of a slide. Take 




Fig. 73. — Proper bottle contain- 
ing sufficient formed feces 
for examination. 




Fig. 74. — Stirring feces in a couple of drops of water on a slide to make 
properly diluted preparation. 



up a quantity of feces about the size of a match head on 
the end of an ordinary wooden toothpick. Stir the feces 
(Fig. 74) in the water on the slide until sufficient has been 



92 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

dissolved off to produce the proper concentration of feces 
for examination. The material should be properly spread 
out on the slide at the same time. Do not let it come to the 
edge or end of the slide (Fig. 72). The solution of feces 
should be about as thick as will just barely permit ordinary 
print to be read through it (Fig. 75). It is not neces- 




Fig. 75. — Proper spread of diluted feces, showing also how print may be just 
read through it. Thicker preparations cannot be examined microscopically 
as satisfactorily. 

sary to dissolve or use all the feces taken up on the pick. 
Return the soiled pick with any undissolved feces on it to 
the bottle or dispose of it in any other convenient way. 
No cover-glass is required. The preparation is ready for 
examination. 

Concentration of ova by means of the centrifuge. — All 
the ova, or at least nearly all of them, in a dram or two 
of feces may be collected by proper use of the centrifuge 
and placed on one side. Considerable concentration of the 
ova (Figs. 76 and 77) may thus be obtained, and whenever 
very small numbers are present this very much facilitates 
the search for them. No examination should be consid- 
ered negative until after this method has been carried out. 
It should be employed, however, only after at least one 
or more slides, prepared in the ordinary way, have been 
examined. 

Add some water to the feces in the bottle. Shake 
until a proper suspension (Fig. 78) is obtained. The 
feces should be diluted about ten times. After the sus- 
pension is of about the proper consistency stop shaking 



FECES 



93 



and do not dissolve up the remaining feces. Now strain 
into a centrifuge tube through two or three layers of 
gauze placed in a small glass funnel (Fig. 79). This 
removes any large coarse particles. Now centrifuge (Fig. 
80) for a sufficient length of time to throw only ova or 
larvse to the bottom of the tube. This depends upon the 
speed of the centrifuge and other factors dependent upon 




Fig. 76. — Photomicrograph of feces prepared by diluting in the ordinary- 
way and showing two tapeworm ova. 

the particular centrifuge used. The length of time re- 
quired by any particular centrifuge may easily be deter- 
mined by experiment with a specimen known to contain a 
good many ova. A good electric centrifuge (Fig. 67) 
requires only about four to eight seconds to throw ova 
to the bottom of the tube. A hand centrifuge (Fig. 69) 
requires longer time, but may be used satisfactorily. 

The diluted feces consists of a suspension of particles, 



94 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 




Fig. 77. — Photomicrograph of the same specimen of feces shown in Fig. 76, 
prepared by centrifuging as directed on page 92. This shows great con- 
centration of the ova, and also the ova of trichuria and ascaris present 
which might be overlooked in examining a preparation not made in this way. 




Fig. 78. — Solution of feces made 
in the bottle in which it was 
brought to the laboratory. 



most of which are much smaller 
than ova. In fact, the bulk of 
normal feces is bacteria. These 
small particles are thrown down 
more slowly than the larger, heav- 
ier ova. It is important, there- 
fore, to run the centrifuge just 
long enough to throw the ova to 
the bottom, and to stop before the 
smaller particles are thrown down. 
Pour off all the contents of the 
tube except the sediment in the 
bottom, which should contain the 
ova. Refill the tube with water to 



FECES 



95 




Fig. T9. — Straining diluted feces into centrifuge tube. 



the former level, shake thor- 
oughly, and centrifuge again. 
Note that the supernatant fluid 
is not so cloudy this time. Pour 
it off, as before. ^Vfter pouring 
off the supernatant fluid the sedi- 
ment may be washed again and 
again until as clear as desired and 
then examined. After pouring off 
the supernatant fluid there re- 
mains the sediment, and at least 
a drop or two of fluid. Shake 
the tube so as to stir up the sedi- 
ment and pour it out on a slide 
(Fig. 71), spreading it properly 
with the test-tube (Fig. 81). It 
is now ready for examination. 

Method of examining a slide 
preparation. — The examination is 




ii 



in 



Fig. 80. 

Tube I — Diluted feces after 
first centrifuging. 

Tube II — After second centri- 
fuging. 

Tube III— After third centri- 
fuging. Note that the su- 
pernatant fluid is clear. 



96 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 



made with the low power objective (16 mm.). The light 
must be adjusted so that objects with little or no color may 
be seen plainly (Fig. 10). Try the effect of opening and 



: -' ,.-v. 



» ■■ 




Fig. 81. — Pouring out and spreading sediment from feces on slide with lip of 

centrifuge tube. 

closing the diaphragm until the best light is obtained. See 
that the light is properly adjusted for each specimen. The 




Fig. 82. — Diagram indicating proper method of examining entire preparation. 
The mechanical stage makes this possible. 

light may require changing from time to time while looking 
at a given specimen. 

Begin at the corner (Fig. 82) of the preparation. By 
means of the mechanical stage move toward the opposite 
end of the slide, looking for ova and larvse as the slide is 




& 



A 








** 









PLATE XII 
INTESTINAL PARASITE OVA AND LARVAE 

A. Uncinaria ova in different stages of development up to larvce. 

B. Uncinaria larva. Note long buccal capsule. 

('. Strongyloides larva. Note short buccal capsule. 

I). Oxyuris ova in different stages of development up to larval stage. 
( )ne is seen on end. 

E. Oxyuris larva. Note long pin like tail. 

I\ Tenia saginata "ova." Those on the right have a thin, delicate cover- 
ing which is frequently seen in fresh preparations. 

(!. Tenia solium "ova." 

II. Hymenolepis nana "ova." 

I. Trichuria trichuris ova. 

J. Ascar.i? lubricoides ova. 



FECES 



97 



moved. Whenever the end of the preparation is reached 
drop down the width of the field and move in the opposite 
direction. Upon reaching the end, drop down the width 
of the field as before, and so continue until the entire prepa- 
ration has been examined. In this way the entire spread of 
material may be looked over in a few minutes. After one 
has acquired experience the slide may be moved quite 
rapidly, only hesitating or stopping now and then to focus 
on and examine more closely some suggestive object. Once 
in a while it may be necessary to turn in place the high 






i ££§& 



Fig. 83. — Photomicrographs of different ova all taken with the same magnifica- 
tion for comparison, a. Uncinaria americana. b. Oxyuris vermicularis. 
c. Trichuria trichuris. d. Tenia saginata. e. Ascaris lumbricoides. 

power objective (4 mm.) to study more closely, under 
higher magnification, some doubtful object, but this lens 
should not be used for routine examination. The field 
covered by this lens is so much smaller that it would take 
entirely too much time to look over a given preparation. 

Uncinaria americana ova, — Uncinaria ova (Plate 
XII, a) are oblong, round-ended, and measure about 38 
microns in diameter and 60 microns in length (Fig. 83). 
They consist of three distinct parts — the shell, the nucleus 
or yolk, and the clear space filled with albumin between 
the yolk and the shell. An object should not be diagnosed 
to be an ovum unless these three separate parts can be 
made out. 

The shell appears as a distinct narrow line. It is regu- 
lar in outline. The nucleus is made up of granular material. 
The granules are probably colorless, but the mass appears 



98 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

gray or dark gray, according to the light used. The nucleus 
usually shows a certain amount of development varying 
from division into two segments up to embryo formation. 
Most ova, however, in fresh specimens are in the two to 
eight segment stage. The nucleolus, a clearer spot, can 
usually be recognized in the segments. The space between 
the shell and the nucleus is of diagnostic importance. The 
nucleus is irregular in outline, according to the stage of 
division, but the shell remains regular. This is in con- 
trast to certain vegetable cells and other objects which 
have an outer membrane and a nucleus, suggesting ova. 
In these objects the outer membrane dips in wherever there 
are indentations in the nucleus, and this gives the object 
an irregular outline. They also vary in size considerably, 
while the size of ova of any given kind of worm is fairly 
uniform. 

Ascaris lumbricoides ova. — The ova of the ascarides 
(Plate XII, J) that infect man vary considerably in size 
and shape. They measure about 64 by 52 microns. The 
outer membrane proper is covered with a thick layer of 
gelatinous material, which is very irregular in outline and 
gives the ovum a very rough nodular appearance. The 
color of this varies from a very light to a very dark brown. 
Ova are sometimes found from which this outer coat has 
been partially or completely torn . 

The nucleus cannot be seen as well as the nucleus of the 
clear ova, largely because of the thick colored outer coating. 
There can, however, always be made out a clear space be- 
tween the shell proper and the nucleus upon proper adjust- 
ment of the light. 

Trichuria trichuris ova. — These ova (Plate XII, I) are 
very characteristic in appearance and should never be mis- 
taken. They are oblong, measure about 52 by 25 microns 
and vary in color from light brown to very dark brown. 
There is a knob of lighter-colored material on either end. 
The nucleus and clear space can be made out in most 
specimens, but the thick, dark-colored shell prevents their 



FECES 99 

being seen as satisfactorily as in the case of some of the 
more transparent ova — uncinaria, for instance. 

Oooyuris vermicularis ova. — These ova (Plate XII, D) 
are flattened on one side and oval on the other. They meas- 
ure about 50 by 30 microns. The shell is thicker than 
that of uncinaria ova, and is shiny in appearance. The 
nucleus and clear albumin space are plainly seen. Empty 
egg-shells are often found. They contain a smaller mass 
of bacteria or debris in place of the nucleus. Larva? (Plate 
XII, E) are sometimes found, and are to be recognized 
by the long, sharp, pinlike tail. 

The eggs of oxyuris are deposited in the rectum and in 
the folds around the anus and vagina. Therefore, much 
larger numbers of eggs can be found in material properly 
collected from these parts than in the feces. A good way 
to collect material for this purpose is to scrape the surface 
of the parts with some suitable instrument, like, for in- 
stance, the end of a microscope slide. Dilute the material 
collected with a drop of water on a slide and examine. 
Both ova and larvae are usually found. 

Hymenolepis nana ova. — These ova (Plate XII, H) 
measure about 48 by 36 microns. They have an outer and 
inner membrane, each of which is quite thin and appears 
as a distinct line. The space between the two membranes 
is clear, except for a few striae, which run irregularly longi- 
tudinally. The nucleus is light gray and contains 4 to 6 
very plain hooklets. The space between the inner mem- 
brane and the nucleus is not very large and looks clear. 

Tenia saginata and tenia solium ova. — These ova (Plate 
XII, F-G) are oval, brownish or grayish colored, and 
measure about 36 by 30 microns. There is an inner and 
an outer membrane, and the space between contains a great 
many radial striations. On account of the color of the 
covering the nucleus is not so well seen. Hooklet-like 
objects are present in both. The ova of both species look 
almost alike. These ova are often seen covered by a vitel- 
line membrane ( Fig. 84 ) . 



100 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

Larvce of uncinaria and strongyloides. — After feces 
containing uncinaria ova have been out of the body for 
twenty-four hours or more some of the ova are likely to 




Fig. 84. — Photomicrograph of ovum of tenia saginata enclosed within its 
vitelline membrane. This membrane is usually soon lost after the 
ova are expelled into the feces, and most specimens do not show it. 

hatch (Fig. 85) if the conditions are favorable. The larvae 
(Plate XII, B) are about 0.21 mm. long by 0.02 mm. 
thick. They wiggle about for a while, but usually die in 
undiluted feces in a few hours. In this stage they are 
known as rhabditiform. They look very much like the 
rhabditiform larva? of strongyloides. 

The ova of strongyloides are deposited by the adult 
worm in the glands of Lieberkuhn, and there hatch, giving 
rise to rhabditiform larva? (Plate XII, C), which are 
passed in the feces. They wiggle about actively, but often 
many of them die after a few hours. They are about the 
same size as uncinaria larvae, and look almost exactly 
like them. 

The most practical differential points between uncinaria 
larvae and strongyloides larvae for one not especially familiar 
with these parasites are: 

1. Whenever uncinaria larvae are present there are also 
many uncinaria ova present. Strongyloides larvae are not 
accompanied by the presence of ova except in cases of 
double infection. 



FECES 



101 




Fig. 85. — Photomicrograph of hookworm Ovum and larva and ovum of trichuria. 

2. Uncinaria larvae are not present in feces under twen- 
ty-four hours after they are passed. Strongyloides larvae 
are present from the first. 

3. The buccal capsule of strongyloides larvae is very 




Fig. 86. — Photomicrograph of anterior end of hookworm larva under high 
magnification. Note long buccal capsule. 



102 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

short, while that of uncinaria larvae (Fig. 86) is rela- 
tively long. 

Examination for amebce in amebic dysentery. — A large 
part of the cases of dysentery occurring in this country, 
and especially those occurring in the tropics, are due to 
pathogenic amebae. There are many species of amebse, only 
a few of which live as parasites upon other animals. They 
are therefore known as endamebae. There is at present 
considerable difference of opinion among authorities on the 
subject, as to whether there are one or more species of 
endamebae found in the intestine of man. It is certain that 
a supposedly harmless amebae — ameba coli — is very fre- 
quently found in the discharges from man, and that this 
species is quite different from the pathogenic species which 
cause dysentery. In view of the present unsettled state of 
the classification and nomenclature relating to the path- 
ogenic amebae, we give methods of diagnosing and differ- 
entiating the pathogenic species from nonpathogenic spe- 
cies only, and do not enter further into the classification. 

Collection of specimens for examination. — It should 
be understood that the disease process of amebic dysentery 
consists chiefly of ulceration of varying degree and varying 
extent in the colon, and that the endamebae are present and 
reproducing in and on the ulcerating tissue. There is more 
or less pus and mucus being thrown off from the diseased 
tissue all the time, and this contains the endamebae which 
we desire to find. During a period of acute active disease 
there may be many discharges daily, consisting chiefly of 
this mucus and pus usually containing more or less blood. 
This is ideal material for examination. Cases of acute 
dysentery can have such actions by making the effort at 
most any time, and furnish the proper fresh specimen for 
examination. In the subacute or quiescent stage there 
may be only an occasional particle of the bloody or puru- 
lent mucus mixed with the fecal stool. The examination 
must be made within one or two hours of the time the 
material was discharged, as endamebae rapidly die and 



FECES 103 

their appearance changes upon exposure to air. During 
cold weather the specimen must be examined even more 
promptly, unless it is kept warm artificially in some way. 
Keeping the bottle containing the specimen in warm water 
is a good way to keep it warm until the laboratory is 
reached. It is always better, whenever practical, to have 
the patient come to the laboratory and there pass the 
stool into a pan or other vessel for immediate examination, 
or bring the microscope to the patient. The next best thing 
is, whenever patients are not near the laboratory, to make 
several proper slides from selected particles of bloody or 
purulent mucus and allow them to dry. These may be 
satisfactorily examined for endamebse at any subsequent 
convenient time by staining them, provided one is at all 
familiar with stained endamebse. 

It should be remarked here that endamebse may be 
demonstrated in purulent material from abscesses of the 
liver and other parts of the body. They are present in 
largest numbers, however, in the walls of such abscesses. 

Examination of unstained material. — With an ordinary 
wood toothpick held in each hand pick out particles of the 
bloody purulent mucus, which probably came from ulcers, 
and place on a slide. Spread just sufficiently to make a 
preparation of proper thickness to examine, but not so thin 
that it will dry out rapidly. By prompt work a proper 
examination can be made before it dries out. It is better 
to spend a short time examining each of several specimens 
prepared from different parts of the stool than a long time 
on one preparation. Those who cannot work rapidly 
enough and those who prefer it, should use cover-glasses. 
Put a cover-glass on the material on the slide and make 
slight pressure, if necessary, to thin out the material, but 
do not make too thin. The high, dry objective is generally 
used to examine for endamebse, but with a little practice 
they can be seen well with the low power lens (16 mm.), 
and the time required to look over a given area is much 
less. The light must be reduced until the hyaline objects 



104 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

on the slide are clearly seen. Pus and blood cells are 
readily recognized. Endamebse (Plate XIII, a) appear 
to be larger cells containing coarse granules and blood 
cells in the endosarc, surrounded by a clear ectosarc. In 
active endamebse the ectosarc is constantly changing its 
shape. It may throw out a pseudopod into which the 
endosarc flows. Another pseudopod may be projected in 
another direction, and either be withdrawn or the ectosarc 
moves into it. These movements (Fig. 87) are slow and 
are known as ameboid motion. The endameba may change 
its position slowly, and finally move out of the field. 

Technic of staining amebce and examination of stained 
specimens. — Many different methods of staining have their 
individual advantages for special purposes. The best, most 
practical method for ordinary diagnostic purposes is the 
carbol-fuchsin and methylene blue stain for pus and exu- 
dates, given on page 140. The ectosarc (outer portion) 
and endosarc (inner portion) are well differentiated (Plate 
XIII, b). The stain varies somewhat, but the ectosarc 
is usually more or less purple, while the endosarc stains 
varying shades of blue. The nucleus is not specially well 
stained by this method, but is well enough stained for 
present purposes. It stains red. The endosarc contains 
from one to several blood cells or fragments of blood 
cells, and usually a few bacteria. Occasionally a very 
small endameba may be found which contains no blood 
cells. Large endameba are two, three or four times the 
diameter of polymorphonuclear neutrophile pus cells, which 
can usually be found present in such preparations for 
comparison. The endamebse found in dysentery average 
considerably larger than those found in alveolodental pyor- 
rhea (endamebse buccalis) (Plate XIII, c). 

Differentiation of pathogenic from non-pathogenic ame- 
bce. — The non-pathogenic amebse found in feces are on the 
average smaller than the pathogenic species, but otherwise 
resemble them very much in their movements and general 
appearance. The striking difference is that the pathogenic 



£Tiif&3i\ 



- - 



/£Wv 









*W- 



-:- 







A. 



ft 



r- 




A. Typical appearance of bloody mucus in amebic dysentery showing active 
endamerjae. High magnification. 

B. Preparation of bloody mucus from a case of amebic dysentery stained with 
carbolfuchsin and methylene blue. Note ingested red blood cells. 

C. Preparation from pyorrhoea lesion stained with carbol-fuchsin and methylene 
blue. Note dark stained inclusion bodies. 




Fig. 87.— Change of shape and position of an endameba during intervals of a 
few seconds each by ameboid motion. 



105 



106 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

species phagocyte blood cells, while the others do not, 
except possibly occasionally. For practical purposes this is 
the best differential point, and the only one necessary. 
Amebse containing blood cells may be considered path- 
ogenic endamebse. 

Interpretation. — In active or acute amebiasis pathogenic 
endamebse can always be found upon proper examination. 
During a quiescent stage of the disease, when few ulcers 
remain and when they are almost healed, it may be impos- 
sible to find endamebae because of the very small number 
present, and the absence of the kind of material in which 
they may be found. It therefore may not be possible to 
determine by a single microscopic examination that a pa- 
tient has not quiescent amebic infection, but it is possible 
to determine that an acute case of dysentery is or is not 
due to endamebse. 

Test for occult blood. — Hemorrhage may occur so high 
in the alimentary tract that the blood is so altered that it 
cannot be recognized in the feces either by macroscopic or 
microscopic examination. Such blood is called occult 
blood. Whenever practical, special precautions should be 
taken to avoid outside sources of blood that would give a 
positive reaction, such for instance as eating food con- 
taining blood, hemorrhages from the mouth and nose, or 
hemorrhoids, etc. A purgative followed by a meat-free 
diet a day or two before the specimen is collected insures 
against blood from food. 

Technic of test. — Make a thick solution of the material 
in water, unless it is already sufficiently liquid. Ordinary 
formed feces would be diluted with about five times as 
much water. Whenever specimens are contained in a 
proper bottle container, the best way to make the solution 
is to add some water in the bottle, replace the stopper, and 
shake until enough of the feces has been dissolved to make 
the proper consistency. It is not necessary that all should 
be dissolved. Place in a test-tube a little benzidine (crys- 
tals). A dip on the point of a knife about half the size 




PLATE XIV 
TEST FOR OCCULT BLOOD 



Tube I. Negative reaction. 
Tube II. Positive reaction. 



FECES 107 

of a pea is ample. Add a few (5 or 6) drops of glacial 
acetic acid. Shake until benzidine is dissolved. Pour in 
about one-half to one c.c. of the diluted feces. Shake to 
mix and then add a few drops (10 or 15) of fresh peroxide 
of hydrogen. It is better to allow this to trickle down the 
side of the tube and overlay the material being tested. If 
occult blood is present, a dark blue ring (Plate XIV) at 
the zone of contact forms within a minute or two. If none 
was present, no blue color appears. If the tube is shaken, 
the whole mixture turns blue, but the ring test is generally 
more striking. 

Interpretation. — A positive test shows the presence of 
occult blood. Occult blood is present in most cases of 
cancer of the stomach and intestines all the time, and 
occasionally in cases of ulcer. Other possible sources of 
blood must be kept in mind in interpreting a positive test. 
A negative test showing the absence of occult blood is 
valuable evidence against the existence of cancer, which is 
the chief value of the test. It is little evidence against 
ulcer, but whenever tests made on several different days are 
all negative, ulcer is usually not present. 



CHAPTER VIII 

PUS AND EXUDATES GENERALLY 

General remarks. — It is often desirable to know what 
kind of bacteria, cells, etc., are present in various kinds of 
material, such as pus, exudates, etc. It is not considered 
within the scope of this book to go into bacteriology except 
to describe those simple methods of investigation that can 
be carried out by those not equipped with a bacteriological 
laboratory, but having the simple comparatively inexpensive 
laboratory equipment suggested in this book. 

There are certain simple tests that may be made which 
enable one to determine in many cases what group of 
organisms bacteria found belong to. One of the distin- 
guishing features of germs is their shape (Fig. 88). We 
have bacilli which are rods of various shapes two or more 
times as long as they are thick, some of which are motile, 
and cocci which are round or oval, the length being less 
than twice the breadth. The cocci are further divided 
according to their arrangement with relation to each other 
into diplococci, arranged in pairs; staphylococci, arranged 
in bunches (grape bunch), and streptococci, arranged in 
chains. Still other classification according to arrangement, 
such as strepto-bacilli, strepto-diplococci, etc., is sometimes 
made. Classification according to morphology is based 
upon stained specimens. 

Making the preparation and staining. — When obtain- 
ing pus or exudates for examination with a view to discov- 
ering the causative germ, it is very important to obtain 
material from the seat of the disease process. For instance, 
it may be that there are many bacteria at the surface of a 
lesion which have nothing to do with the cause of it, but are 

108 




Fig. 88. — Drawing illustrating morphological classification of bacteria. 

a. Staphylococci. b. Streptococci, 

c. Diplococci. d. Bacilli, 

e. Streptobacilli. f. Diphtheroid bacilli. 

g. Spirillae or curved bacilli. h. Spirochete. 

109 



110 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 



simply growing secondarily in the pus and exudate. These 
bacteria may be much more numerous in the material at 
the surface, while in the diseased tissue none are to be 
found. The material should be properly spread upon a 
slide (see Fig. 89) and allowed to dry. Whenever it is 



* ..*r~ 



Fig. 89. — Proper spread of pus or similar material to be stained and examined. 
There are thin and thick areas affording any thickness desired. 

desirable to examine a very fluid material for bacteria, it 
should be centrifugalized to concentrate the bacteria and 
cells. The supernatant fluid is poured and drained off 




Fig. 90. — Thoroughly draining sediment in bottom of centrifuge tube. 
Every drop of fluid is poured off. 

thoroughly (Fig. 90) and some of the sediment spread upon 
a slide with a platinum loop. 

The best routine stain for such preparations is the 
carbol-fuchsin and methylene-blue stain (for technic, see 
page 137). To determine the presence or absence of acid- 
fast bacteria, use the carbol-fuchsin, acid and methylene- 
blue stain (for technic, see page 115). To determine the 
reaction of the bacteria to Gram's stain, use Gram's method 
(for technic, see page 138). 



PUS AND EXUDATES GENERALLY 111 

To determine whether bacteria are motile or non-motile, 
put a drop or less of the fluid material on a slide; or, if the 
material is too thick, dilute some of it on a slide with normal 
salt solution. No cover-glass is required except in rare 
instances. Examine with the "high dry" lens with reduced 
light. The beginner often mistakes Brownian motion for 
actual motility. In Brownian motion the objects appear 
to be dancing and moving actively; but, barring the influ- 
ence of currents in the fluid, "they do not move from place 
to place. On the other hand, motile bacteria do move 
(swim about) from place to place. It is good practice to 
examine a few old specimens of urine for bacteria. 

Application in practice. — By making the simple tests 
of morphology, motility and staining reaction, one often 
gains valuable information as to what class of bacteria are 
present and, what may actually be of more value, what 
bacteria are not present. Below we give a short list of 
common bacteria, with their staining reaction, morphology 
and motility. 



CHAPTER IX 

SPUTUM 

Examination for tubercle bacilli. — The practical labora- 
tory diagnosis of pulmonary tuberculosis rests upon the 
demonstration of acid-fast bacilli in the sputum. There are 
several other acid-fast bacilli that resemble tubercle bacilli 
morphologically and otherwise, and therefore the laboratory 
diagnosis is not absolute. It is true that the identity of the 
bacilli could be determined by sufficient laboratory investi- 
gation, but the time and facilities required prevent it from 
being practical for ordinary purposes. 

Collecting specimens. — Many a failure to find tubercle 
bacilli in cases of tuberculosis has resulted from the exam- 
ination of improper specimens, such as saliva or secretion 
from the posterior nares containing no sputum from the 
lungs whatever. At the time specimens are requested for 
examination the patient should be informed that material 
that comes up from the lungs is wanted, and not saliva 
and nasal secretion. A mixture of several expectorations is 
desirable. 

Most patients raise more sputum in the early morning, 
after a period of sleep and rest, than at any other time of 
day. Such sputum usually contains more tubercle bacilli 
than that raised at other times of day. 

A clean, wide-mouth bottle, with new cork stopper, 
makes an appropriate container. Unless the container is 
furnished or specified, specimens are likely to be brought in 
the most inappropriate things, such as pasteboard boxes, 
small tin boxes, etc. Too small a quantity for the most 
thorough examination is likely to be brought, unless the 
patient is instructed. 

Owing to chemical and bacteriological changes that 

112 











Name 


Morphology 


M 


ii. 


1. Staphylococcus albus. 


Small, oval or round organisms; usually in clusters. 




in 


2. Staphylococcus aureus. 


Small, oval or round organisms; usually in clusters. 




m 


3. Staphylococcus citreus. 


Small, oval or round organisms; usually in clusters. 




Di 


4. Streptococcus pyogenes. 


Small, oval or round organisms; usually found in 
chains. 




ne 


5. Streptococcus mucosus. 


Large, oval organism, encapsulated; in chains. 




ne 


6. Streptococcus pneumoniae (pneumococcus). 


Slightly elongated cocci in pairs, surrounded by cap- 
sule. Often in chains. 




111 


7. Micrococcus meningitidis (meningococcus). 


Bean-shaped cocci, in pairs. 




m 


8. Micrococcus gonorrhoeae ( gonococcus ) . 


Bean-shaped cocci, in pairs. 




M 


9. Micrococcus catarrhalis. 


Bean-shaped cocci, in pairs. 




IK 


10. Micrococcus tetragena. 


Bean-shaped cocci, usually occurring in fours. Large 
size. 




IIP 


11. Bacillus anthracis (anthrax bacillus). 


Large rods, single or in chains. 


IK 


12. Bacillus subtilis. 


Large rods, single or in chains. 




111 


13. Bacillus diphtheria? ( Kleb's-Loffler-bacillus ) . 


Beaded or granular rods, or clubs. 




ie 


14. Bacillus pseudo-diphtheria*. 


Beaded or granular rods, or clubs. 




die 


15. Bacillus coli (colon bacillus). 


Short rods. 




ill 


16. Bacillus enteritidis. 


Short rods. 




iv 


17. Bacillus typhosus (typhoid bacillus). 


Short rods. 




h 


IS. Bacillus paratyphosus (paratyphoid A or 
B). 


Short rods. 


h 


19. Bacillus dysenteriae. 


Short rods. 




He 


20. Bacillus mucosus capsulatus. 


Short rods., capsule. 





ie 


21. Bacillus pestis (plague bacillus). 


Short, plump rods, with a clear center occasionally. 


ie 


22. Bacillus of "pink eye" (Koch-Weeks 
bacillus) . 


Small, thin rods. 




In 1 . 


23. Bacillus tetani (tetanus bacillus). 


Long, slender rods, with one end knobbed. 


;•' 


24. Bacillus pertussis. 


Small, short oval rods. 




ie 


25. Boas-Opeler bacillus. 


Large, oval rods. 




:e, 


26. Bacillus pyocyaneus. 


Small, slender rods — varying in size — often chains. 




ie. 


27. Bacillus chancer oideus (bacillus of 
Ducrey ) . 


Long, thin rods. 


if. 


28. Bacillus tuberculosis (tubercle bacillus). 


Slender, often slightly curved, rods. 




ie 


29. Bacillus smegmse (smegma bacillus). 


Slender, often slightly curved, rods. 




ie 


30. Bacillus [butter or grass]. 


Slender, often slightly curved, rods. 


if. 


31. Bacillu3 leprae (Hansen's bacillus). 


Slender, often slightly curved, rods. 




i 


32. Spirillum c'holerae (cholera vibrio) . 


Short, slightly twisted rods, or comma-shaped. 


1 




p. 











LITY 


Staining 
Reaction 


Where Organisms May Usually be Demonstrated 


ie. 


Gram -\- 


Pus from local infections. Normal skin. 


le. 


Gram + 


Pus from local infections. Xormal skin. 


ie. 


Gram -f- 


Pus from local infections. Xormal skin. 


ie. 


Gram + 


Virulent local infections, showing spread via the lymphatics. 


ie. 


Gram -f- 


Respiratory mucous membrane. 


ie. 


Gram + 


Respiratory mucous membrane, and especially in sputum from lobar pneumonia. 


ie. 


Gram — - 


Spinal fluid in cerebro-spinal meningitis. 


ie. 


Gram — 


Pus from gonorrheal urethritis, vaginitis or ophthalmia. 


ie. 


Gram — 


Respiratory or vaginal mucous membrane. 


ie. 


Gram -f- 


Respiratory or vaginal mucous membrane. 


ie. 


Gram -f- 


Blood of anthrax (charbon) patients. 


ie. 


Gram + 


Frequent contamination of blood, media, urine, etc. 


ie. 


Gram -f- 


Suitable cultures from diphtheritic lesions in respiratory tract. 


;e. 


Gram -J- 


Respiratory mucous membrane. 


;ht. 


Gram — 


Urine, feces, sputum, etc. 


ive. 


Gram — 


Urine, feces, sputum, etc. 


ive. 


Gram — 


Suitable cultures from blood, urine and feces of typhoid patients. 


ive. 


Gram — 


Suitable cultures from blood, urine and feces of patients with para-typhoid 
fever. 


ie. 


Gram — 


Suitable cultures from feces of patients with bacillary dysentery. 


e. 


Gram -f- 


Respiratory mucous membrane. 


te. 


Gram — 


Enlarged lymph glands, or suitable cultures from blood in plague. 


"• 


Gram — 


Conjunctival pu3 in ''pink-eye." 


e. 


Gram + 


The primary lesion of tetanus (lockjaw). 


€ - 


Gram — 


In sputum from patients with pertussis (whooping-cough i . 


e. 


Gram — 


Stomach contents in reduction or absence of HC1. 


e. 


Gram — 


"Blue-pus" infection of wounds. 


. 


Gram — 


Pus from non-syphilitic ulcerative conditions of genitalia (chanceroides) . 


e. 


Acid fast. 


Sputum, or pus, from lesions of tuberculosis. 


e. 


Acid fast. 


Material collected from under the male foreskin, or female genitalia. 


e. 


Acid fast. 


Butter, greasy foods and fresh vegetables. 


e. 


Acid fast. 


Scraping from the nose, or nodules, or lesions of leprous patients. 


e. 


Gram — 


Suitable cultures from feces of cholera patients. 









SPUTUM 113 

take place in sputum upon standing, the characteristic 
staining reactions by which tubercle bacilli are recognized 
are frequently altered. . In some specimens the bacilli begin 
to lose their staining characteristics in twenty-four to forty- 
eight hours. Specimens of sputum, therefore, should not 
be more than twenty-four hours old — the fresher the 
better. 

Making the preparation. — Tubercle bacilli are not pres- 
ent in all parts of a specimen of sputum. It is therefore 
necessary to make the preparation for examination from the 
kind of material most likely to contain bacilli. Many a 
failure to find tubercle bacilli in specimens containing them 
has resulted from examination of preparations made of 
material fished out of a bottle, jar or other container with 
a platinum loop or other improper instrument. Pour out 
a proper quantity of the sputum into a Petri dish (we do 
not know of anything else as good). It is an advantage 
to set this on the table and place something (the cover of 
the dish) under one edge so as to tilt it and allow the 
sputum to flow to one side, leaving the remaining portion 
of the bottom uncovered by sputum. This furnishes a 
convenient surface onto which particles may be drawn and 
teased out in order to separate the desirable from the un- 
desirable material. Ordinary wood toothpicks are best 
for this purpose, as well as to spread the material upon the 
slide with. Use two picks (Fig. 91), one in each hand, 
and do not try to pick out material for examination with 
only one. 

Select j the gray, purulent particles to be seen in the 
sputum. With the picks drag them onto the uncovered part 
of the bottom of the dish. Tease and separate from adher- 
ing mucus, saliva, froth, etc. Transfer to a slide. Get out 
several other particles from other parts of the sputum mass, 
mix and spread all out on the slide. Thus is made a prepa- 
ration representing favorable-looking material from several 
different parts of the sputum and much more likely to con- 



114 PKACTICAL CLINICAL LABORATORY DIAGNOSIS 

tain tubercle bacilli than one of similar size made from any 
one part of the sputum. 

The spread of material on the slide should be compara- 
tively large, but it should never reach the ends or edges of 
the slide. Burn the contaminated ends of picks and discard. 

The preparation must now be allowed to dry. There is 
no harm in warming the bottom side of the slide to hasten 
drying. It is not necessary that the very thick masses 




Fig. 91. — Picking out favorable material from sputum in a Petri dish. 
Note use of two toothpicks. 

sometimes present on such preparations should be perfectly 
dry before staining, as these are not examined. 

Staining. — Tubercle bacilli are surrounded by a fatty 
capsule or covering. This is what gives them their most 
characteristic staining property known as acid-fast. This 
fatty covering is not readily penetrated by ordinary stains, 
and it is also resistant to alcohol and mineral acids. By 
employing a mordant with the stain and by the aid of heat 
the bacilli can be stained within their fatty covering. The 
mordant usually employed is carbolic acid. When once 
stained, tubercle bacilli are not decolorized by mineral acids 
like other bacilli that have no fatty covering, because the 
acid cannot penetrate the covering. This distinguishes 
tubercle bacilli from most other bacilli and the different 
steps in the staining technic have this object in view. 



SPUTUM 



115 



The different steps are as follows: 

1. Fix with heat. 

2. Stain with carbol-fuchsin plus heat one minute. 

3. Decolorize with mineral acid. Wash. 

4. Counterstain with Loffler's methylene blue. Wash, 
dry, and examine with oil immersion lens. 

The fixation is accomplished by passing the slide slowly, 
film side up, through the flame of a Bunsen burner or 




M 

Fig. 92. — Touching the heated slide to the back of the hand to judge the tempera- 
ture and avoid getting it hot enough to damage the film of material to be 
examined when fixing with heat. 

alcohol lamp two or three times. Do not get the slide 
hotter than it can be borne on the back of the hand (Fig. 
92 ) . Use a very small flame. Hold the slide in the hand 
when fixing it. 

There are several formula? for making the carbol-fuchsin 
stain. None are better than Czaplewsky's. It keeps in- 
definitely and some others do not. When ordering, specify 
carbol-fuchsin, Czaplewsky's formula. The formula is: 

Basic fuchsin 1 gm. 

Carbolic acid, liquefied 5 c.c. 

Neutral glycerine 50 c.c. 

Water to make 100 c.c. 



116 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

The ingredients are added and mixed in the order given. 
The fuchsin can be purchased in original 10 gramme bottles 
and the stain can be made up much more cheaply than the 
price at which the staining solution usually sells.- How- 
ever, one ounce of the staining solution, if properly used, 
is sufficient to stain more than one hundred specimens, and 
it may be found more convenient to buy the prepared stain 
in many instances. 

This, like the other stains, should be kept in a proper 
drop bottle for use. Hold the slide by the edges near the 
end, between the thumb and first finger of one hand, and 
put on the stain with, the other hand. Allow one drop of 




Fig. 93. — Proper way to apply stain to a slide. Spread the stain over the 
material to be stained as it flows from the drop bottle. 

the stain to flow on the film and by means of the lip of the 
drop bottle spread (Fig. 93) it over the film, allowing 
more stain to flow out of the bottle as needed. A quan- 
tity equal to two, or at the most three, drops is sufficient 
for any slide. The stain should not be spread to the edge 
of the slide. It is extremely awkward to flood the slide 
with stain so that it runs off on the hands, table, etc. 

After the stain has been placed on the slide it should 
he heated by passing it slowly through the flame two or 
three times. It must not be boiled. Just let it get 
hot is sufficient. Forceps or special slide-holders are not 
necessary to hold the slide with while heating ( Fig. 94 ) . 
Anybody who can't heat such a slide properly while hold- 
ing it with his fingers without burning his fingers or spill- 
ing the stain about, needs laboratory instruction or practice 



SPUTUM 117 

and not a pair of forceps. After heating, the slide may be 
placed on the slide-rest over the sink or waste jar for a 
minute, or it may be held in the hand. 

We use a two and one-half per cent solution of sulphuric 
acid to decolorize with. Weaker solutions decolorize more 
slowly. Much stronger solutions are more likely to alter 
other bacteria and cells present in the preparation. The 
acid solution may be made by placing 2.5 c.c. of sulphuric 
acid in a measuring cylinder and adding sufficient water to 
make 100 c.c. 

Pour the stain off of the slide and drop on several drops 




Fig. 94. — Heating slide in staining with carbol fuehsin. Slide holders are 
unnecessary. Use a small flame and small amount of stain. 

of the acid solution. Allow the first few drops to run off 
(Fig. 95), and wash off the excess of stain that remained 
on the slide. Note that the red color of the film fades and 
in less than a minute becomes a very light pink or purple 
and ceases to fade more. It is now decolorized. There is 
no necessity to continue the decolorization process, but no 
harm would result from the application of this strength 
acid solution for several minutes. By this decolorization 
the stain is taken out of all other bacteria, cells, etc., but if 
tubercle bacilli or other acid-fast bacteria are present they 
retain the stain. The slide is washed by running water 
over it. 



118 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 



This completes the staining process so far as the tuber- 
cle bacilli are concerned, but the red stained bacilli show up 
better against a contrast-stained background, and it often 
is desirable to have other bacteria and cells on the slide 
stained so they can be seen at the same time. Loffler's 
methylene blue is an excellent counterstain for this purpose. 
It should be put on and spread over the film in the same 




Fig. 95. — Decolorizing with sulphuric acid solution. The slide is held so that 
the fluid flows over the preparation and off at the end or corner. 



way as the carbol-fuchsin. It usually stains sufficiently in 
less than one-half minute. Now wash well. Wipe the 
back, ends and sides of the slide dry. Take up any excess 
of water by "blotting" with the towel (or paper) and dry 
by warming it over the flame. Don't get it too hot. 

No cover-glass is required. Place some immersion oil 
on the slide and examine with the oil immersion lens for 
the red-stained tubercle bacilli. 



Plate XV. 



■:. 









- 



• ■:: 



I 

A 



£ 






gj 



' \ 



mm A 



B. 




*IF 






#$ 



j *^i 



& 



M 















■ 






'fc^m. 



C. 




,<•*% 



A. and B. Tubercle bacilli in sputum stained with car,bol-fuchsin-acid- 
methylene blue stain. Note greater amount of beading in B than in A. 

Lepra bacilli in material scraped from leprous lesion. Carbol-fuchsin- 
acid-methylene blue stain. Note the large number of bacilli present and the 
characteristic lepra cells filled with the bacilti. 

D. Pneumococci in sputum. Simple carbol-fuchsin and methylene blue 
stain. Note lanceolate shaped diplococci. 



SPUTUM 119 

Description of tubercle bacilli. — The bacilli (Plate XV, 
A-B) may be present in large numbers and in almost every 
field, or they may be so few that one or more are found 
only after several minutes' search. There often are clumps 
of several bacilli, but usually the number is small and 
many are found separate from others. It is common to see 
two or more bacilli lying parallel to or somewhat across 
each other. Two may be seen lying end to end. Most 
tubercle bacilli are slightly curved, and in many specimens 
they are slightly beaded in appearance. 

Interpretation. — It seldom occurs that any acid-fast 
bacilli are found in the sputum except tubercle bacilli. For 
all practical purposes acid-fast bacilli found in sputum, 
in the presence of clinical evidence of tuberculosis, are 
tubercle bacilli. A thorough examination of a proper 
specimen of sputum without finding any acid-fast bacilli 
is evidence that the patient has not tuberculosis advanced 
to the stage in which there is breaking down of lung tissue. 
It is not, however, proof of this, since it often occurs that 
bacilli are found only after repeated examinations. It is 
not possible, therefore, to determine with certainty that a 
patient has not tuberculosis by not finding tubercle bacilli, 
no matter how thorough the examination. 



CHAPTER X 

LEPROSY 



Technic of obtaining material and making preparations 
for examination. — Lepra bacilli are present in the tissues 
of leprous lesions, and in the case of tubercular leprosy 
they are almost incredibly numerous. Since the bacilli are 




Fig. 96. — Scraping leprous lesion. Bleeding is prevented by pressure of 

the fingers. 



chiefly in the fixed tissue cells, it is necessary to scrape up 
the tissue and examine the scrapings for the bacilli. Most 
lepra lesions are anesthetic, and no pain is produced. Catch 
up and squeeze (Fig. 96) the suspected tissue between the 
thumb and finger. With a scalpel scrape down into the 
tissue and secure scraped-up tissue fairly free from blood. 
This is accomplished by keeping the area anemic by the 
pressure of the fingers. The edge of the lesion often con- 
tains the most bacilli. When tubercles are present, scrape 
them. Spread the scraped material on a slide and allow 
to dry 

120 



LEPROSY 121 

Staining. — Lepra bacilli are acid fast and are to be 
stained exactly like tubercle bacilli (see page 114). 

Appearance of lepra bacilli. — Lepra bacilli (Plate XV, 
c) look like tubercle bacilli, to ordinary observation at least, 
but in most specimens the very much larger numbers are 
striking. Many of the bacilli are seen arranged in masses 
or in the so-called lepra cells. There is no staining or 
morphological difference between lepra bacilli and the tuber- 
cle bacilli that permits differentiating them. 

Interpretation. — Large numbers of acid-fast bacilli, es- 
pecially if grouped like lepra bacilli, found in material 
scraped from tissues showing clinical evidence of leprosy, 
are for practical purposes lepra bacilli. The absence of 
acid-fast bacilli in scrapings of a tissue indicates very 
strongly, but does not prove infallibly, that the tissue is 
not leprous. 

Lepra bacilli are almost always found in the nodular 
type and ulcerative lesions. The anesthetic leprous ery- 
themas on the cutaneous surfaces are sometimes due to lo- 
calization of bacilli along the nerve trunks and the lesion 
itself may not show anv bacilli. 



CHAPTER XI 

SPINAL FLUID 



Meningitis. — The different forms of cerebro-spinal men- 
ingitis can usually be easily diagnosed by proper microscopic 
examination of cerebro-spinal fluid obtained by lumbar 
puncture. Since we now have a valuable specific remedy 




Fig. 97. — Introducing the needle in making lumbar puncture with patient in 
sitting position. The skin has been sterilized at the site of puncture with 
tincture of iodine. 



for at least one form (meningococcic), exact diagnosis be- 
comes of great importance. 

Obtaining material and making pre paration. — It is not 
considered within the scope of this book to describe the 
technic of lumbar puncture (Fig. 97). We would state, 
however, that it is a very simple operation, and easy to 
perform. The patient should be turned from side to side 
a few minutes before the puncture is made, in order to 

122 



SPINAL FLUID 



123 



stir up the cells in the canal, which tend to settle to the 
lower side when the patient lies in one position for some 
time. The quantity of fluid drawn for diagnostic purposes 
should be 10 c.c. It should be allowed to drop (Fig. 98) 




Fig. 98. — The spinal fluid is allowed to drop directly into the centrifuge tube. 



directly from the lumbar puncture needle into two clean 
test-tubes or small bottles. The first one is likely to con- 
tain some blood cells. Therefore, the second one should be 
used for examination, especially in making cell counts and 
globulin tests. The sooner the examination is made after 
the fluid is drawn the better, except in the case of tubercu- 
lar meningitis, in which it is better to wait two or three hours 
for the pellicle of fibrin to form. 

Sometimes the fluid is very cloudy and purulent, in 
which case smears should be made at once when it is drawn. 
When not quite cloudy it is necessary to centrifugalize the 



124 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

specimen, in order to concentrate the cells and bacteria. 
Centrifugalize long enough to thoroughly settle the sus- 
pended cells, etc., to the bottom. Pour off the supernatant 
clear fluid and drain well (Fig. 90). With platinum loop 
make proper spreads of the sediment on slides. A spread 
about one-quarter of an inch wide and one inch or more 
long is satisfactory. Allow these to dry in the air and then 
stain. Usually a pellicle of fibrin forms in fluid from tu- 
bercular meningitis upon standing for an hour or two. As 
this contracts it gathers tubercle bacilli in its meshes, if any 
are present. This should be "fished" out and spread upon 
a slide or slides for examination. If no pellicle forms a cen- 
trifugalized specimen should be examined. A high speed 
centrifuge will throw down tubercle bacilli fairly well. 

The best stain for all kinds of pus is the carbol-fuchsin 
and methylene-blue stain (see page 137 for the technic). 
It is usually desirable to make a Gram's stain also (see 
page 138 for the technic). Whenever tubercle bacilli are 
to be looked for, the carbol-fuchsin, acid and methylene- 
blue stain is to be employed (see page 114 for the technic). 

Meningococci. — Meningococci (Plate XVI) are intra- 
cellular (and extracellular) coffee-bean-shaped diplococci. 
In some specimens they are quite numerous, but in many 
others they are not. Frequently a search of many minutes 
is required to find a few pairs of cocci. It is a good idea 
not to diagnose as meningococci any organism that is not 
intracellular. The meningococcus is Gram negative. 

Pneinnococci. — In pneumococcal meningitis the pneu- 
mococci (Plate XVI) are usually very numerous. They 
are chiefly extracellular, but many are intracellular. The 
lanceolate-shaped diplococcus, which is Gram positive, is 
quite characteristic. 

Tubercle bacilli. — Tubercle bacilli from the cerebro- 
spinal fluid (Plate XVI) stain and look like the same or- 
ganism from other sources. They are usually not numerous. 

Other bacteria. — Other bacteria of various species may 
be, in rare instances, the cause of meningitis, and may be 



Plate XVI. 




Diplococcus Intracellularis. 
Menincococci 



ftp 



*** 



Qs 




r 




$ 



■ 






# 



Bacillus Influenza. 



#•* 



.*V 



>* 



dim 




» ■** 



Diplococcus Pneumoniae. 
Pneumococci 



!«i ; 








Bacillus Tuberculosis. 



Preparations of cerebrospinal fluid from meningitis due to different bacteria. 
All specimens stained with carbol-fuchsin and methylene blue except the one containing 
tubercle bacilli which is stained with carbol-fuchsin decolorized with acid and counter- 
stained with methylene blue. 



SPINAL FLUID 125 

found in the cerebrospinal fluid. This may occur especially 
in traumatic meningitis. 

Cell counts. — To make a total cell count in spinal fluid 
make a preparation in the Bass counting chamber, count 
the cells as in counting leucocytes in blood and multiply the 
number counted on the ruling by 2.5. This gives the num- 
ber per cubic millimeter. Usually no dilution is necessary 
in counting the cells in those fluids in which cell counts are 
required — tuberculosis; poliomyelitis and syphilis, especially. 
If there are too many cells to count in an undiluted speci- 
men, the fluid may be suitably diluted with Toison's fluid 
or salt solution, in which case the count must be multiplied 
by the dilution and also 2.5 to get the total number per 
cubic millimeter in the undiluted fluid. 

To make a differential cell count, drain off the super- 
natant fluid and make a spread on a slide of the cell sedi- 
ment collected by centrifugalization. Stain either with 
Wright's stain or with the carhol-fuchsin and methylene- 
blue stain, and count as* in counting blood. 

Test for globulin increase. — Put saturated aqueous so- 
lution of ammonium sulphate in a test-tube to a depth of 
about one inch. Overlay this carefully with spinal fluid 
which has been cleared of cells by centrifuging. A good 
way is to allow the fluid to flow down the side of the tube 
from a pipette. An increase of globulin is shown by a ring 
varying from a very faint whitish ring appearing not later 
than one hour to a very heavy ring appearing immediately. 

Interpretation. — The presence of meningitis and the 
causative organism can usually be diagnosed with satisfac- 
tion by proper microscopic examination. It sometimes 
happens that there are so few meningococci or tubercle 
bacilli present that these organisms may not be found. 
When no pus cells are present there is no meningitis pro- 
duced by pyogenic organisms. When many pus cells are 
present and no bacteria found, the case is usually meningo- 
coccal meningitis. 

Normal spinal fluid contains up to 10 cells per cubic 



126 PKACTICAL CLINICAL LABORATORY DIAGNOSIS 

millimeter, and these are all mononuclear cells. The count 
in poliomyelitis usually runs 100 to 300 cells with 5% to 
10% neutrophiles. In tubercular meningitis the count usu- 
ally runs 300 to 1000 cells and approximately 100% mo- 
nonuclears. In tabes there are usually 90 to 300 cells per 
cubic millimeter, and in paresis about 30 to 90, practically 
all of which are mononuclears. In meningitis due to any 
pyogenic organism the total count is enormously increased, 
nearly all the cells being neutrophiles. 

Increase of globulin indicates some meningeal infection 
or disease, including poliomyelitis and syphilis. 



CHAPTER XII 

DIPHTHERIA 

Principles of laboratory diagnosis of diphtheria. — It is 
possible to make a practical diagnosis of diphtheria bacilli 
in the laboratory by reason of the fact that when grown 
upon certain culture media (Loffler's blood serum) they 
grow much more rapidly than the other bacteria with 
which they are associated in the nose, mouth and throat, 




Fig. 99.— Bass diphtheria culture tuhe. The tube on the left has been broken 
open and the swab has been straightened out ready for use. 

and that on this media they show characteristic mor- 
phology not shown by any other bacilli likely to be found 
in these places. 

Material required. — A tube of Loffler's blood serum 
and a sterile swab are required. Tubes and swabs are 
furnished by city and state board of health laboratories, 
but they soon deteriorate. A special hermetically sealed 
tube containing the swab (Fig. 99) also was devised by 

127 



128 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

one of us (Bass) and is to be preferred especially by those 
who have to buy their culture tubes. This tube is small, 
convenient, and keeps indefinitely. One doing general or 
special practice of medicine who is likely to see cases of 
probable diphtheria should include one or more of these 
tubes in his armamentarium. Any doctor equipped with 




Fig. 100. — Removing cotton plug from culture tube preparatory to inoculating 

with the swab. 



a microscope and not having access to a general bacterio- 
logical laboratory should keep such tubes on hand. 

Making the culture. — In cases where diphtheria is sus- 
pected there often is membrane or other local evidence of 
the disease, either on the tonsils, uvula, fauces or posterior 
nares. Often, however, no membrane is recognized, but 
still diphtheria is suspected and a laboratory examination 
for diphtheria bacilli is desired. It is desirable to obtain 
material to be planted on the culture media from the sur- 
face of the diseased mucous membrane, and as free as 



DIPHTHEKIA 129 

possible from bacteria and secretions from any other part. 
Most people who are old enough to try can show the 
pharynx without the tongue depressor being used, by open- 
ing the mouth wide and making a gagging effort. At this 
moment the swab should be rubbed over the surface of 
the diseased part. In the case of a young child use of 
the tongue depressor is necessary. In nasal diphtheria the 




Fig. 101. — Inoculating culture media by rubbing swab over surface. 
Note how cotton plug is held. 

swab should be passed into the nose and some of the secre- 
tion secured. Now, holding the culture tube in the left 
hand and the swab in the right, remove the cotton plug 
from the tube (Fig. 100), rub the swab over the surface 
(Fig. 101) of the culture media gently and then replace 
the plug. The best way to dispose of the infected swab 
is to throw it in the fire if one is convenient, or it may 
be returned to the empty end of the tube and all wrapped 
in paper to be destroyed at a more convenient time. 



130 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

In spite of the duty the physician owes to his patient to 
be prepared for such work as this whenever it is indicated, 
it often happens that a culture tube is not at hand when 
the suspicious case is first seen. Instead of waiting until 
the next visit to get a culture tube, an extemporaneous swab 
may be made by winding a little absorbent cotton on a suit- 
able stick and taking the culture with it. The inoculated 
swab is placed in a dry, clean, small, empty bottle or test- 
tube or in an envelope, and is carried to the office or labora- 
tory, where a culture tube is inoculated in the way described 
above. 

Incubation. — The inoculated tube or culture, which is 
usually made at the bedside of the patient, should be 
started to incubating at once. No special laboratory facili- 
ties or incubator are required. The tube should be wrapped 
with paper to insure against the plug coming out and 
placed in an inside (vest) pocket where the heat of the 
body keeps it warm enough to favor the growth of diph- 
theria bacilli. This is as good as an incubator. The tube 
may be kept warm in the pajama pocket during -the night. 
Incubation is necessary, because the bacilli grow very slowly 
and have not their characteristic morphology unless the 
culture is kept warm. This method of incubation is espe- 
cially advantageous because it starts from the time the cul- 
ture is first taken and it may permit the diagnosis to be made 
several hours earlier. 

Examination of culture. — Whenever many bacilli are 
present they usually have grown to sufficient numbers in 
six or eight hours and have their characteristic morphology 
so that they may be recognized upon examination. No visi- 
ble growth will be present at this time. If the exigencies 
of the case demand very early diagnosis the culture may 
be examined after six hours and very often the diagnosis 
can be made. We have occasionally been able to make 
the diagnosis in four hours. If no diphtheria bacilli 
can be found upon early examination, the incubation should 



DIPHTHERIA 131 

be continued and subsequent examination made. An ex- 
amination should be made after eighteen to twenty-four 
hours incubation before a final negative diagnosis is made. 
Whenever very few bacilli are present it may require this 
long for sufficient bacilli to grow for them to be found. 
After twenty-four hours other bacteria which may be 
present often overgrow the diphtheria bacilli and prevent 
satisfactory diagnosis. It is therefore desirable to make 
the final examination by the expiration of this period. 

Making preparations for examination. — A good plati- 
num loop is very valuable in examining the culture. Often 
the loop of the wire is too large, not smooth, or other- 
wise improper. The loop can be properly formed and 
shaped by bending the end of the platinum wire snugly 
one time around the end of a smoothly sharpened lead 
pencil just where the lead disappears under the wood. 
After the loop has been formed, bend it back sharply in 
order to bring the center of the loop in line with the 
straight wire. Once a good loop is provided, take care not 
to bend it out of shape, as many do. 

The growth on the culture media is not fluid enough 
to spread well on a slide and it is an advantage to dilute 
it (on the slide) with some water. Place approximately 
the smallest quantity of water that the platinum loop will 
hold near the middle of the side. Now hold the culture in 
the left hand and the platinum loop handle in the right. 
Sterilize the loop (Fig. 102) by heating red (white) hot in 
the Bunsen-burner or alcohol flame. Remove the cotton 
plug (Fig. 103) from the tube and draw the loop over the 
surface (Fig. 104) of the culture, endeavoring to scrape 
off any growth present. Often little or no growth can 
be seen. Stir the material removed from the culture in 
the water on the slide and spread out in a thin long streak 
(Fig. 105). Too thick smears are not good. Now flame 
the loop again and replace the cotton plug. As soon as 
the preparation dries it is ready to be stained. 



132 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 




± 



Fig. 102. — Sterilizing the platinum loop in flame of Bunsen burner. 
Note proper size and shape of loop. 




Fig. 103. — Removing cotton plug, sterile platinum loop held in hand. 



DIPHTHERIA 



133 



Staining. — The steps in staining are: 

1. Fix with heat. 

2. Cover the entire spread with Loffler's methylene blue 




Fig. 104. — Scraping the surface of the culture with the platinum loop to 
obtain bacteria for examination. 

and allow to stain approximately one-half to one minute. 
Wash, dry, and examine with oil immersion lens. 



Fig. 105. — Proper long thin streak of material from the culture to be 
stained and examined. 



To fix, pass through the flame slowly, film side up, 
two or three times. Don't get it hotter than can be borne 
on the back of the hand ( Fig. 92 ) . 



134 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

Loffler's methylene blue is a valuable routine laboratory 
stain. The formula calls for: 

Saturated alcoholic solution of methylene blue 

(Griibler's) 30 c.c. 

Solution of potassium hydroxide, 1 to 10,000 100 c.c. 

Mix. 

If the American made methylene blue is used, the fol- 
lowing formula is better : 

Methylene blue 0.5 gm. 

Grain alcohol 30 c.c. 

Solution of potassium hydroxide, 1 to 10,000. . . .100 c.c. 
Mix. 

A good way to make up the alcoholic solution of 
methylene blue is to put 10 grams of methylene blue in 
a 100 c.c. (4 oz.) bottle and nearly fill with alcohol (95%). 
Shake thoroughly and allow to settle. This keeps, as does 
also the staining solution. After removing a part of the 
saturated alcoholic solution of methylene blue for use the 
bottle may be refilled with alcohol and more solution will 
be ready for use again. 

Sometimes it may be desirable to stain suspected diph- 
theria preparations with Gram's stain also in doubtful cases. 
See page 138 for the technic. Diphtheria bacilli are Gram 
positive, and the darker staining of the polar bodies shows 
these up well. 

Description of Diphtheria Bacilli 

When taken directly from the seat of disease or when 
grown on ordinary media diphtheria bacilli cannot be differ- 
entiated by their morphology from many other species of 
bacilli likely to be found present in the mouth, nose and 
throat ; but when grown on the Loffler's blood serum media 
they present a morphology quite characteristic of them 
and a small group of so-called diphtheroid bacilli (Plate 



Plate XVII. 



* 
















V 


* 


- V 








t rf> 






/ 




V 

\ 


^-^ 


* - 


>/ • 












I 






*• 


2* 






a 






/ 


^ 


% v - « ^k 


h>- 






■ 1 






> 


1 


\x 



( 






'•■ r 



i 



Diphtheria bacilli from different cases suggesting variation in morphology. 
All of the specimens were stained with Loffler's methylene blue. One was counter- 
stained with safranin. Note that the polar bodies are shown better. 



DIPHTHERIA 135 

XVII). In the first place they are bacilli and no other 
organism found need be considered as possibly diphtheria. 
They vary in their size from small up to very large. Some 
will be iive or ten times longer or larger than others. Many 
are club-shaped, others are more or less lanceolate-shaped 
on one or both ends. Most of the diphtheria bacilli are 
more or less beaded and in many this is so marked as to 
make the bacilli look somewhat like chains of streptococci. 
In addition to the beaded condition a variable proportion 
of the bacilli contain one, two, three or four much darker 
staining granules, called polar bodies. When only one or 
two are present in a bacillus they are usually situated near 
its ends. These polar bodies are much more numerous in 
some specimens than in others. They are fairly well shown 
with the methylene blue stain, but they are shown much 
better by counterstaining with a red stain such as safranin 
or carbol-fuchsin (Plate XVII, D). After staining the 
whole preparation with Loffler's methylene blue, stain one 
end with 1% safranin solution one-half minute, or with 
carbol-fuchsin on and off; wash, dry and examine with oil 
immersion lens. It is seldom that any other diphtheroid 
organisms show these polar bodies and this is a fairly re- 
liable differential point, but not infallible. 

In most cases cultures that contain diphtheria bacilli 
contain other bacteria, cocci and possibly bacilli. Pure cul- 
tures are seldom obtained from diphtheria lesions. 

Interpretation. — Diphtheroid bacilli, Gram positive and 
in the presence of clinical evidence of diphtheria are for all 
practical purposes diphtheria bacilli. A properly taken cul- 
ture properly incubated and examined always shows diph- 
theria bacilli in the presence of the disease. In the absence 
of any clinical evidence whatever one should look with doubt 
upon diphtheroid bacilli. 



CHAPTER XIII 

GONORRHOEA 

Obtaining material and making preparation. — During 
acute gonorrhoea there is a great deal of pus formed, con- 
taining many gonococci. If the patient is a male the pus 
should be wiped from the meatus in order to get rid of 
the other bacteria that may be growing in the pus after 
it is exposed to external contamination. A fresh drop is 
brought to the meatus by "stripping" along the urethra. 
When there is very little pus being formed this is espe- 
cially necessary. The fresh pus squeezed from the follicles 
in the urethra is much more desirable than old pus that has 
been standing under the prepuce or even in the urethra. 
When the urethritis is far back, of course it is necessary 
to "strip" from far back. In chronic gonorrhoea where 
there is very little pus formed and most of that in the pos- 
terior urethra, seminal vesicles and prostate, this pus can 
usually be obtained by massaging and forcing it forward 
with the finger through the rectum. 

In acute gonorrhoea in females the external pus should 
be removed from the orifice of the vagina and meatus, and 
fresh pus obtained from higher up. There are many other 
bacteria in the outer pus and gonococci often cannot be 
demonstrated in it with satisfaction. The best material for 
examination for gonococci in acute cases is obtained by in- 
troducing a finger into the vagina and "stripping" the 
urethra forward. In chronic cases pus may be obtained from 
the cervical canal or glands through a vaginal speculum. 

Sometimes it may be desirable to examine the urine for 
gonococci which are present in gonorrhoeal cystitis. Re- 
cently voided urine is absolutely necessary. Centrifuge the 

136 



GOXORRHCEA 137 

urine until the pus cells are thrown to the bottom of the tube. 
If not enough sediment is collected the supernatant urine can 
be poured off and the tube refilled and centrifuged again. 
This may be repeated several times if necessary, until suffi- 
cient pus sediment is secured. Pour off all the supernatant 
urine and drain well to get rid of all urine. By exercising 
care the last drop can be drained off, leaving thick pus in 
the bottom of the tube. With a platinum loop or other 
suitable instrument make a proper spread of this on a slide. 
A spread approximately one-fourth inch wide and one and 
one-half inches long is ideal. The proper thickness can be 
learned with a little experience. The ideal spread is only 
one cell thick and the cells should be well separated and 
not all packed together. All pus should be spread with a 
single stroke to avoid damaging the cells. 

Carbol-fuchsin and methylene blue stain. — The best 
routine stain for gonorrhoeal or any other kind of pus or 
exudate is a combined stain, carbol-fuchsin and mythylene 
blue. It shows up the cells and bacteria to the greatest 
advantage. The steps are: 

1. Fix the dry film with heat. 

2. Cover film with carbol-fuchsin for a few seconds. 
Wash. 

3. Cover film with Loffler's methylene blue, ^ to 1/2 
minute. Wash, dry, and examine with oil immersion lens. 

To fix, pass the slide, film side up, slowly through the 
flame of a Bunsen burner or alcohol lamp two or three times. 
Do not heat any more than can be borne to touch the back 
of the hand. 

The stain should cover the film only and not the entire 
slide. The best way is to touch the dropper of the drop 
bottle, with a drop of stain on it, to the film and spread 
(Fig. 96) the stain over the film as it is allowed to flow out 
of the bottle. 

Gram's staining method. — Many of the bacteria that 
may be mistaken for gonococci are stained by Gram's method 
while the gonococcus is not stained by this method and there- 



138 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

fore is called Gram negative. Gram's stain does not show 
up the morphology of bacteria as well as the carbol-fuchsin 
and methylene blue stain does, and therefore is to be used 
largely for further test or source of evidence. Gram's stain 
should be made only after the other has been made and 
intracellular diplococci have been found. In most cases the 
picture is so definite that in practice the Gram stain is not 
required. 

The technic of Gram's stain as we do it is: 

1. Fix with heat. 

2. Stain film with carbol-gentian violet about two 
minutes. 

3. Pour off carbol-gentian violet and cover film with 
Gram's solution one minute. 

4. Decolorize with alcohol. Wash. 

5. Counterstain with one per cent, safranin solution 
one-half minute. Wash, dry and examine with the oil 
immersion lens. 

The carbol-gentian violet should be made according to 
Czaplewsky's formula, which is: 

Gentian violet 1 gram. 

Liquefied carbolic acid 5 c.c. 

Neutral glycerin 50 c.c. 

Water to make 100 c.c. 

Mix. This stain keeps indefinitely. 

Instead of washing off the carbol-gentian violet with 
water the excess should be washed off with a few drops 
of Gram's solution. The formula for Gram's iodine solu- 
tion is: 

Iodine 1 gram. 

Potassium iodide 2 grams. 

Water to make 300 c.c. 

Mix. This solution keeps well. 

Alcohol from 95 to 100% is all right to decolorize with. 
Hold the slide by one end. Let the other end be lower and 



GOXOKRHCEA 



139 



drop (Fig. 106) alcohol on the upper end of the film so that 
it runs over the film and off at the lower end of the slide. 
Only a dozen or two drops are necessary to decolorize. 
Whenever decolorization is complete it will be noted that 
the drops of alcohol as they run off are not colored as was 
the case before. The alcohol is washed off to facilitate the 




Fig. 106. — Decolorizing with alcohol. The slide is held so that the alcohol which 
is dropped on it flows over the film and off at the end. Tilting the slide 
from side to side facilitates the decolorization. 



counterstaining with an aqueous stain which does not mix 
well with the alcohol. 

If we should examine the preparation under the micro- 
scope after decolorization we would find all Gram positive 
bacteria stained a very deep violet and the Gram negative 
bacteria unstained. Unstained bacteria are difficult to see 
and we therefore stain with a stain that contrasts well with 
the violet. The unstained objects take the counterstain 
and are now fairly readily seen. Of the several available 



140 PKACTICAL CLINICAL LABORATORY DIAGNOSIS 

counterstains we choose a one per cent, aqueous solution of 
safranin.. The formula is : 

Safranin 1 gram. 

Water to make 100 c.c. 

Mix. This stain keeps well. 

Appearance of gonococci. — Gonococci are readily phago- 
cyted by the polymorphonuclear leucocytes and therefore 
a large portion of those present in pus are within the pus 
cells (Plate XVIII) . There are no other bacteria for which 
gonococci are likely to be mistaken that are found intra- 
cellular to the same extent. Though some gonococci are 
to be found outside of cells it is best not to call anything a 
gonococcus that is not intracellular. The number in one cell 
varies from one to twenty or thirty or more pairs of cocci. 
The cells packed with diplococci found in acute gonorrhoea 
are so typical that one who is familiar with them could 
hardly make a mistake. Many of the pus cells have no 
gonococci in them. In chronic gonorrhoea one may look 
over many fields of the microscope before he finds a pus cell 
with gonococci in it, while in acute gonorrhoea practically 
every field has them in it. 

With the carbol-fuchsin and methylene blue stain gono- 
cocci stain blue, while with the Gram stain they take the red 
counterstain. 

Gonococci are always in pairs — true diplococci. The 
division between the two coffee-bean shaped organisms can 
be made out in most of them, but of course in some they 
are not seen in the proper position for the division to be 
made out. 

Interpretation. — Intracellular, Gram negative, diplo- 
cocci from the urethra are for practical purposes gonococci, 
especially when typically arranged in the cells. In the ab- 
sence of such diplococci an acute urethritis is not gonor- 
rhoea. It is not possible to determine beyond doubt that 
gonorrhoea (chronic) is not present in a given case, but thor- 
ough, repeated, negative examinations are strong evidence 
that it is not. 



Plate XVIII. 






.'8 




A. 




c. 



i 




It 




^ 









PP 










B. 



ii& » K* 



D. 






A. Pus in acute gonorrhoea. Carbol-fuchsin and methylene blue stain 
-K. Fus in acute gonorrhoea. Gram's stain. 

C. Pus from case of acute non-specific urethritis. Many staphylococci 
present. Carbol-fuchsin and methylene blue stain. 

D. Pus obtained from prostate and seminal vesicles by massage in case of 
chronic gonorrhoea showing a few gonococci. Carbol-fuchsin and methyl 



blue stain. 



ene 



CHAPTER XIV 

SYPHILIS 

I. Examination for Treponema pallida, — There are two 
simple practical methods of demonstrating Treponema pal- 
lida: with the darkfleld microscope and by preparing with 
India ink. The former is far superior to the latter, but re- 
quires from about fifteen to thirty dollars worth of extra 
apparatus. There is hardly any question, however, but that 
those who assume the responsibility of the diagnosis of 
syphilis are under solemn obligation to their patients to the 
extent of either being prepared to make such examinations 
or to have them done by others who are prepared to do so. 
The diagnosis by inspection of early lesions of syphilis often 
remains in doubt, but it can almost always be made with 
certainty by proper microscopic examination. 

Obtaining material and making preparation for examina- 
tion with the darkfield condenser. — The Treponema pallida 
is present usually in very large numbers in all chancres, 
mucous patches, condylomata, adjacent swollen lymph nodes 
and in smaller numbers in other syphilitic skin lesions and 
most other syphilitic lesions of the body. It must be under- 
stood that the organisms are in the tissue and not on it. In 
order to obtain material containing them, we must obtain 
it from the diseased tissue. In the case, for instance, of a 
chancre, it is necessary to obtain material from the hard 
syphilitic tissue and not simply from the indurated or ul- 
cerated tissue over or around it. What is needed is "juice" 
from the diseased tissue and the scraped up tissue itself. It 
is very necessary to avoid getting a large amount of blood 
with the material. To obtain proper material (Fig. 107) 
grasp the chancre between the thumb and forefinger and 

141 



142 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

make sufficient pressure to drive out the blood, as is shown 
by the anemic appearance of the tissue. Maintain the pres- 
sure and with a scalpel scrape down into the hard chancre 
tissue. Scrape up some of the tissue and squeeze out some 
"juice" of the tissue. Either transfer these to a slide with 
the scalpel or touch the slide to the drop of fluid that has 
been squeezed out. Sometimes patients will not endure the 
pain produced. In case of an ulcerated lesion a drop of a 
one per cent, cocaine solution may be applied to the ulcer 
to anesthetize it, or infiltrate the tissue beneath the chancre 
with a one-half per cent, solution of cocaine. In the case of 




Fig. 107. — Scraping chancre. Sufficient pressure is maintained to render the 

tissue anemic. 

skin lesions on any part of the body the same technic is 
followed. Whenever lesions in the mouth are so located on 
the lip or tongue that they can be manipulated in the same 
way, it should be done. Otherwise, it is better to pinch off 
a little of the diseased tissue with a suitable instrument, 
rinse it in salt solution and then to crush it on a slide with 
the end of another slide and obtain proper fluid from the 
diseased tissue. The diagnosis is less dependable whenever 
the lesion is in the mouth on account of the possibility of 
finding the nonpathogenic spirochetes commonly present in 
the mouth and mistaking them for Treponema pallida. If 
the scraped-up material is too thick it may be diluted with 
a little salt solution. Cover the small quantity (about one- 
fourth drop) of " juice" from the diseased tissue with a 



SYPHILIS 



143 



cover-glass and it is now ready for examination with the 
darkfield condenser microscope. 

Examination with the darkfield condenser. — There are 
several makes of darkfield condensers on the market. Some 




Fig. 108. — Best form of darkfield condenser. It fits in place of the Abbe 
condenser of the make of microscope for which it is intended. 

are placed on top of the stage of the microscope and 
are interchangeable or usable on any microscope. Others, 
which are the best (Fig. 108), are made for each particular 
make of microscope and fit in the sleeve in the place of the 




'%^J:k 





Fig. 109. — Diagram showing path of rays through a darkfield condenser, and a 
3/52 inch oil immersion lens fitted with funnel stop. 

Abbe condenser. In the use of any make of darkfield 
condenser the Abbe condenser must be slipped out or swung 
out, according to the make of microscope being used. 

The principle (Fig. 109) of darkground illumination is 
that by means of a central stop no direct light is permitted 



144 



PKACTICAL CLINICAL LABORATORY DIAGNOSIS 



to pass through the object, while by means of a system of 
lenses and reflectors the object is illuminated by strong light 
reflected from the sides and at an angle. In this way the 
particles suspended in the fluid are shown as very bright 
objects against a dark or black background. 

A very strong light is necessary. Direct sunlight is per- 
fect when available. Those who do not have many of such 
examinations to make, and those who have not electric cur- 
rent, may find it a fairly satisfactory source of light. In 




Fig. 110. — Gas-filled Mazda lamp for dark-ground illumination. May be screwed 

in ordinary light socket. 



most instances a time for the examination can be set when 
the sun is shining. 

Where electric current is available and considerable use 
of this method of examination is made a gas-filled Mazda 
lamp with concentrated filament (Fig. 110) or a small arc 
lamp will be found more satisfactory. 

Slides and cover-glasses of proper thickness should be 
used. If the manufacturers have not stated the require- 
ments for the instrument you have with the general direc- 
tions write them for the information, or better, for a supply 
of a box of slides and cover-glasses suitable for your ap- 
paratus. 



SYPHILIS 



145 



To examine the specimen, place a drop of immersion oil 
(or water) on the darkfleld condenser which must have 
previously been centered. This is done by moving it with 
the set-screws while observing the small ring in its center, 
under the low power of the microscope. The examination 
may be made with the high dry lens, but the treponemas 
appear quite small under this magnification. The oil im- 
mersion lens gives the most beautiful field, but is a little 




Fig. 111. — Illustration of the position of the funnel stop placed in the lens case 
of y 12 inch oil immersion lens for darkfleld work. 



more difficult to use. It must have a funnel stop (Fig. 
111). The makers will furnish the stop for a few cents. 
Stops are already supplied with some microscopes. 

There is a great deal of Brownian movement of all the 
small particles suspended in the fluid under examination and 
we now see many things that cannot be seen by direct illu- 
mination. The suspended particles have more or less dancing 
motion. Spirochetes (Fig. 112) are seen in living, active 
state. They apparently revolve in corkscrew fashion and 
also move laterally. They have little locomotion but are 
carried about more or less by currents. One should practice 
on preparations made from material taken from between the 
teeth which usually contains more or less spirochetes of other 
species. 



146 



PKACTICAL CLINICAL LABORATORY DIAGNOSIS 



India ink preparation. — Only a small portion of the 
spirochetes present can be seen in an India ink preparation. 
It is important to have a good quality of India ink. The 
Gunther Wagner brand is a good one. The "juice" from 
the suspected tissue is mixed on a side with about an equal 




Fig. 113. — Photomicrograph of Treponema pallida in "chancre juice," as seen 
with the darkfield microscope. 

amount of India ink, and spread upon the slide. A wood 
toothpick is convenient to mix and spread with. A little ex- 
perience is necessary to learn just what thickness to make 
the preparation, but it is a good idea to make some thin 
and some thick areas (Fig. 113). Practice with material 
scraped from the edge of your own gums between the teeth, 




Fig. 113. — Proper India ink preparation. Note variation in thickness of spread. 



which generally contains the Spirocheta Dentimn. Allow 
the preparation to dry and examine with the oil immersion 
lens, using strong light. Spirochetes (Fig. 114) appear as 
perfectly clear spirals, against the black granular back- 
ground. Other objects, such as bacteria, blood cells, etc., 
are also shown. 




Fig. 114. — Photomicrographs of India ink preparations containing spirochetes. 

a. Treponema pallida. Compare the red blood cells. 

b. Treponema pallida. c. Spirocheta refringens. 
d. Treponema microdentium and Treponema macrodentium. 

147 



148 PRACTICAL CLINICAL LABORATORY DIAGNOSIS 

Differentiation between Treponema pallida and other 
spirochetes. — We hardly think it wise for most observers to 
undertake to differentiate Treponema pallida from some of 
the other spirochetes upon morphological differences. 

There are spirochetes in the mouth, nose, throat, rectum 
and vagina of a large per cent, of all persons, but there are 
no non-pathogenic species on the skin. Spirochetes found 
in scrapings from lesions not of mucous membranes are 
therefore most certainly either the pallida or, in the very 
rare tropical disease, yaws, the Treponema pertenuis. 
Spirochetes found in material from lesions of mucous mem- 
branes are quite likely to be the common species found in 
the orifices of the body. If, however, the precaution is 
taken to thoroughly cleanse the surface before scraping, 
these are less likely to be encountered. 

The Treponema pallida have from about six to ten or 
more turns, while most of the other spirochetes have fewer. 
The pallida is small and the turns are short. There are five 
or more turns to the diameter of an erythrocyte. This is a 
convenient (though not absolute) test, because in most speci- 
mens we have a few erythrocytes to which any spirochetes 
found may be compared. 

Gland puncture. — The enlarged glands, inguinal and 
others, in early syphilis usually contain very many trepo- 
nemas, and gland puncture usually furnishes a little fluid 
very rich in them. The puncture is a very simple operation. 
A good hypodermic syringe and No. 24 needle are needed. 
The syringe and needle should be dry. Sterilize the skin 
over the intended site of puncture with tincture of iodine 
(an area one-fourth to one-half inch in diameter is large 
enough) and try to carry the point of the needle into the 
center of the gland. Make suction and rotate the needle a 
little. Stop the suction and withdraw the needle. Make 
preparations of the gland "juice" and examine in the same 
way as material obtained from other sources. 

Interpretation. — Small spirochetes from a gland punc- 
ture or from a lesion not on a mucous membrane are for 



SYPHILIS 149 

practical purposes Treponema pallida and practically make 
a diagnosis of syphilis. A competent examination of a sus- 
pected lesion with the darkfield condenser for treponemas, 
if negative, is almost proof that it is not syphilis, but it is 
not infallible proof to this effect. 

II. Wassermann serum test. — It is not within the scope 
of this book to enter into the fundamental principles in- 
volved in the serum test for syphilis. We do not describe 
the original Wassermann technic. Those who desire to use 
this and to understand the principles of the test are referred 
to books on serology. We will, however, describe a simple, 
modified method of making the complement fixation test 
for syphilis which can be carried out by persons with very 
little laboratory equipment, and without much experience 
in this kind of work. We will give the exact technic, every 
step, but will not undertake to explain the reason for every- 
thing that is done. 

Materials required. — (1) We require an alcoholic solu- 
tion of antigen. This is kept as stock and a small amount 
is diluted with salt solution whenever a test is to be made. 
Antigen may be obtained from commercial sources. The 
dose must be known. It must be known how much it must 
be diluted, so that 0.2 c.c. contains the proper dose when the 
hemolytic unit is 0.1 c.c. of 12%% washed guinea pig's 
blood and the amount of active human serum necessary to 
hemolize this quantity of cells. 

The source of supply may furnish this information or 
we may work it out ourselves. All that is necessary is to 
make tests with known positive and known negative bloods, 
employing different quantities of antigen solution. In this 
way we may determine the smallest quantity that will give 
positive reactions with positive blood, and the largest quan- 
tity that will give negative reactions with negative blood. 
Usually there is a good margin between them, and we "split" 
the difference. 

A good antigen will require to be diluted about fifty 
to two hundred times. At present, antigen with the dose 



150 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 



stated can also be obtained from Dr. F. M. Johns, P. O. 
Box 770, New Orleans, La., for $2.00 in advance for suffi- 
cient to make about one hundred tests. 

(2) Washed guinea-pig blood cells, 12%%. Etherize 




Fig. 115. — Etherizing guinea-pig preparing to draw blood. Cup with cotton to 
hold ether makes the best "cone" for this purpose. 



a guinea-pig (Fig. 115). Thrust the needle attached to an 
all-glass syringe directly into the pig's heart (Fig. 116). 
Dissecting one or two previously will serve best to tell where 
the heart is located. The needle should be not larger than 




Fig. 116. — Introducing the needle while steadying the parts. 



SYPHILIS 151 

No. 24. It is not necessary to have the needle and syringe 
sterile. Draw 1 c.c. of blood (Fig. 117). Put this in a 
test-tube with 6 or 8 c.c. of salt solution. Centrifuge until 
the cells are collected at the bottom. Pour off the super- 
natant fluid and refill to make 8 c.c. This is called washed 




Fig. 117. — Drawing blood from guinea-pig. 

guinea-pig cells, 12%%. It should be made fresh for each 
day's work. 

(3) Salt solution, 0.9%. Dissolve 9 gm. sodium chloride 
in 1,000 c.c. water. It is convenient to keep this in a bottle 
with tubing, pinch-cock, etc., arranged on a shelf like the 
water bottle outfit used in staining slides (Fig. 130). 

(4) Patient's blood to be tested. We collect from 5 to 
10 c.c. for this purpose from a vein usually at the bend of 
the elbow. The best apparatus to draw it with is an abso- 
lutely dry, all-glass syringe (Fig. 118) and Xo. 24 platino- 
iridium needle. It is not necessary that they should be steril- 
ized. Sterilize the needle just before use by mopping it with 
tincture of iodine. Blood can be drawn with the needle 
alone by inserting it into the distended vein and allowing 



152 PKACTICAL CLINICAL LABORATORY DIAGNOSIS 

the blood to flow into a test-tube, but this is not so satis- 
factory. With the patient sitting, or if in bed lying, and 
the operator sitting in proper position, have patient grasp 




Fig. 118. — Bur- 
roughs, Well- 
come all -glass 
syringe with 
platino - iridium 
needle attached. 
There are other 
good and cheaper 
all-glass syringes 
of the Luer type 
on the market 
now. 




Fig. 119. — Drawing blood from patient. Arm 
hanging down. Patient grasping above the 



Hanging, uuwu. j. auciu gia 

elbow to distend the veins. 



the arm above the elbow (Fig. 119). This is better than 
tying something about the arm. If held properly, this 
prevents the return flow of blood which distends the veins. 
Have him clinch the fist (Fig. 120) of the arm to be bled. 
This further distends the veins. If still not distended suffi- 
ciently, you can force more bipod into them by gripping 
(Fig. 121) the arm just below the elbow. Now apply 
tincture of iodine to an area about the size of a silver dime 



SYPHILIS 



153 



over the point selected to stick the needle in. With a finger 
of the left hand pull (Fig. 122) the skin tight over the vein 
and, holding the syringe in the right hand, which is steadied 




Fig. 120. — Further distention of veins by clinching the fist. Iodine has been 
applied to the skin over the vein selected for puncture. Note the small 
area. 

against the arm of the patient, stick the needle at the proper 
angle into the vein. By deliberation and accuracy it is easy 
to strike the vein almost every time. Do not stick through. 




Fig. 121. — Squeezing the arm to further distend the veins. 

Now draw (Fig. 123) the desired quantity of blood. Take 
care not to pull the needle out or stick it through the vein 
while drawing the blood. 

Instruct patient to "let go" his grip. Then quickly 
withdraw the needle and make pressure (Fig. 124) over 



154 PKACTICAL CLINICAL LABORATORY DIAGNOSIS 



r 

% 




Fig. 122. — Introducing the needle. Note proper angle of needle and manner of 
steadying the hand. Note also how the skin is pulled tight over the vein 
and in the opposite direction to the stick of the needle. 



. jf 




Fig. 123. — Drawing the blood. 




Fig. 124. — Making pressure over puncture to prevent bleeding under skin. 



SYPHILIS 155 

the puncture for a minute or two with a corner of a towel 
or piece of gauze. This prevents subcutaneous hemorrhage 
of a drop or two that otherwise often occurs. 

Now remove the needle from the syringe and force the 
blood into a clean, dry test-tube before it has time to clot 
in the syringe. Do not use a syringe that is also used for 
giving medicines by injection if avoidable. 

Remove the iodine stain from the arm of the patient 
with a few drops of alcohol. Clean up the syringe before 
the blood clots in the needle. 

The blood should be tested in from two to twenty-four 
hours after it is drawn. It is not necessary to keep it on 
ice if it will be tested within twenty-four hours. 

Such specimens may be sent to other laboratories by 
mail for examination when you are not prepared to make 
the test yourself. Stopper the tube with a new cork, pack 
carefully in a mailing case, and send by special delivery, 
first class, sealed mail. Do not register or send by parcel 
post. Do not send to arrive on a Sunday or a holiday for 
fear of delay in delivery. 

Apparatus required. — (1) All-glass syringe, 5 (or 10) 
c.c, with a one inch platino-iridium needle No. 22 for taking 
blood from patient and a 2 c.c. all-glass syringe with No. 24 
platino-iridium needle for bleeding guinea-pigs. These 
needles can be sterilized just before using by mopping with 




Fig. 125.— Wood test-tube block 2x4x8 inches. Holes may be bored to 
accommodate different size tubes. 

tincture of iodine. They are quite soft, and therefore are 
likely to get dull, but can be resharpened on a small fine- 
grained stone. 



156 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 



(2) Two or three dozen plain lip less test-tubes, 12 x 115 
mm. outside measure, should be provided. These are the 
same kind of tubes used in the other work described in this 
book. They are adopted for purposes of uniformity. 

(3) Two (one would do) graduated pipettes, 1 c.c. 
graduated in hundredths. The graduation should not come 
to the end or tip. 

(4) Test-tube rack, double row, bored to accommodate 
two rows of six of these tubes each. See Fig. 127. 

A block of wood 2x4x8, with a double row of holes 
bored one inch deep in it, makes a splendid rack (Fig. 125). 




Fig. 126. — Small electric incubator suitable for making complement fixation tests 
and other laboratory work by one who does not have more elaborate equipment. 



An incubator is convenient because the reaction is more 
active at 98° F. A small electric incubator (Fig. 126) is 
suitable, and they are now comparatively inexpensive. 



SYPHILIS 



157 



METHOD OF MAKING THE TEST 

1. Set up in the rack two rows of five tubes each (Fig. 
127). 

2. Having previously centrifuged the patient's blood in 
order to separate the serum from the clot, now place in the 




Fig. 127. — Showing arrangement of tubes in rack and use of 1 c.c. pipette in 
measuring serum, antigen, etc., into tubes. Note end of pipette touching 
side of tube. 



first tube of the first row 0.03 c.c. of serum. In the second 
put 0.04 c.c. In the third put 0.05 c.c. In the fourth put 
0.06 c.c. In the fifth put 0.07 c.c. In measuring small 
quantities into tubes from a pipette allow the end of the 
pipette to touch the side of the tube (Fig. 127). Place the 
same amounts in the corresponding tubes of the second row. 

3. Put 0.2 c.c. salt solution in each tube in the first row. 

4. Put 0.2 c.c. diluted antigen in each tube in the second 
row. The stock alcoholic solution of antigen has already 



158 



PRACTICAL CLINICAL LABORATORY DIAGNOSIS 



been diluted with salt solution sufficiently that 0.2 c.c. con- 
tains the proper dose of antigen. 

The dilution would he made by placing a small quantity 
of antigen (say 0.05 c.c.) in the necessary amount of salt 
solution. 




Fig. 128. — Proper method of washing pipette with saline solution from a water 
bottle equipped with tube and pinch-cock. 



We now have duplicate rows of tubes containing gradu- 
ally increasing quantities of the patient's serum. Those of 
the first or control row contain no antigen, while those of 
the second or test row contain antigen. Shake them so as 
to mix their contents. Allow them to stand five to ten 
minutes if kept in an incubator at 98.6° F., or double that 



nnnnn r 


test 


























A 






&■ 




I 'pi n n n n 




I' f 




















D 


















| 














































■ 










- 




































*fl 



PLATE XIX 
MODIFIED COMPLEMENT FIXATION TEST FOR SYPHILIS 

A. Negative reaction. 

B. Negative reaction. 

C. Doubtful positive reaction. 

D. Positive reaction. 

E. Strong positive reaction. 



SYPHILIS 



159 



length of time if kept at a warm living room temperature. 
5. Place in each tube 0.1 c.c. of the washed guinea-pig 
cells. It is important to shake the tube containing the sus- 
pension of cells, because they tend to settle to the bottom. 



CONTROL 





Tube 




1 

0.03 c,c. 
0.2 

o.'i 


2 


3 


•4 


5 


Pt. Serum 

Saline solution 

Guinea-pig cells. . . . 


0.04 c.c. 
0.2 

6'i'" 


0.05 c.c. 
0.2 

6:i" 


0.06 c.c. 
0.2 

6!i 


0.07 c.c. 

0.2 

6!i 



TEST 





Tube 




1 


2 


3 


4 


5 


Pt. Serum 

Antigen solution . . . 


0.03 c.c. 
0.2 

6!i 




0.04 c.c. 
0.2 

o'.i 


0.05 c.c. 
0.2 

6!i" 


0.06 c.c. 
0.2 

o'l 


0.07 c 
0.2 


c. 


Guinea-pig cells. . . . 


0.1 







Fig. 129. — Scheme showing distribution of serum, antigen, etc., in the control 
tubes and the test-tubes. Dotted lines represent incubation. 



Shake the tubes gently to mix and allow to incubate or 
stand ten to twenty minutes (Fig. 129). 

6. Read the reaction (Plate XIX) . In the control tubes 
it will be noted that hemolysis has taken place except per- 
haps in the first, or first and second, or possibly in the first, 
second and third tubes. Hemolysis is indicated by the cloudy 
suspension of cells changing to a clear solution. One or 
more of the tubes will usually show partial hemolysis. In 
case of a negative (non-syphilitic) serum the tubes of the 
test row will show practically exactly the same amount of 



160 PKACTICAL CLINICAL LABORATORY DIAGNOSIS 

hemolysis as in the control. A slight amount of anti- 
hemolytic influence is exerted by the antigen, however, and 
we therefore read the reaction in the test-tube next above 
the lowest control tube in which hemolysis has taken place. 
If, for instance, tube number three is the lowest in which 
complete hemolysis has occurred in the control series, we 
read the reaction in the fourth tube of the test series. If 
hemolysis is complete the reaction is negative, but if hemol- 
ysis is slight or not present, then the reaction is positive. 
The stronger the reaction the larger will be the number of 
tubes in the test row in which hemolysis is partial or absent. 
If no hemolysis is present in the first tube above, and those 
above it show some hemolysis, we would call it positive. If 
there is no hemolysis in the first two tubes above, we would 
call it strong positive. A certain amount of experience and 
judgment are, of course, necessary in this as in other tests 
of this nature. There will be some doubtful reactions, as 
occurs with other serum tests. When in doubt, give the 
patient the benefit of the doubt, according to the clinical 
evidence. 

The modified Wassermann test described above is not 
applicable to spinal fluid because of absence of native com- 
plement. For technic and interpretation of the globulin 
test and cell counts in syphilis, see Chapter X. 

Interpretation. — The above method of making the test 
is as reliable as the original or Wassermann, when done by 
competent and careful laboratory workers and with reliable 
antigen. It is so simple that it can be done with a consid- 
erable degree of satisfaction by many who are not familiar 
with the technic and principles of the original Wassermann. 

Practically all cases of active syphilis after the appear- 
ance of the secondaries give positive Wassermann reactions. 
Practically all non-syphilitic persons give negative reac- 
tions. As the disease improves under treatment or other- 
wise, or as it becomes chronic, there is less and less chance 
of the blood giving a positive reaction. If the reaction is 
positive it is weaker. Many cases of tertiary syphilis and 



SYPHILIS 161 

uncured syphilis give negative reactions. On account of 
the fact that there are some syphilitics who give negative 
reactions and an occasional non-syphilitic who gives a posi- 
tive reaction, the test cannot be relied upon for infallible 
diagnosis of the disease or for the contrary diagnosis. It 
must be considered in connection with the clinical evidence 
in the case for it to be most valuable. When thus considered 
it is often of much value. 

The test is usually negative before the appearance of 
secondaries and should not be allowed to take the place of 
the much more reliable examination for Treponema pallida. 



APPENDIX 

LIST OF APPARATUS AND MATERIAL 
REQUIRED 

1. Microscope complete with mechanical stage attached, 

Figs. 1, 2 and 5. 

2. Dark field condenser if diagnosis of suspected syphilitic 

lesions is to be made, Figs. 108, 109 and 111. 

3. Gas filled Mazda lamp if darkfleld work is to be done. 

The No. 1782 lamp with rheostat for 110 volt D. C. 
or A. C. circuits sold by Bausch & Lomb Optical Co., 
is recommended. Fig. 110. 

4. Mazda lamp, 25 watt, round frosted globe, if electricity 

is available and you care to use this somewhat more 
satisfactory source of light. See p. 6 and Fig. 5. 

5. 1 "blood sticker," p. 12, Fig. 11. 

6. 1 (or 2) box of 50 microscope slides, medium thick- 

ness, white glass, ground edges. Sometimes slides 
are sold that have more or less discoloration in the 
center. If not perfectly clear throughout, don't 
accept them. 

7. 1 box of 100 cover-glass, 7 /g in. square, No. 2. Some- 

times cover-glasses are sold that are cloudy in the 
center. Don't accept them. 

8. 100 c.c. grain alcohol (95%). 

9. 1 tube (6 tablets) Burroughs, Wellcome & Co. "soloid" 

tablets for making Wright's stain. This is sufficient 
to make 90 c.c. of Wright's stain. Make up only 
30 c.c. at a time. See p. 22. 
10. 100 c.c. Merck's methyl alcohol, highest purity, to make 
Wright's stain. See p. 10. You can economize in 
the end by buying an original package of 500 c.c. 
bottle. If kept tightly corked it keeps indefinitely. 

163 



164 



APPENDIX 



11. 1 half -gallon water bottle, tubing and Mohr's pinch 
cock, arranged as shown in Fig. 130. A piece of 
glass tubing is so shaped that one end may be car- 
ried to the bottom of the bottle, while the other 
turns down on the outside of the bottle. Three or 




Fig. 130. — Arrangement of work table where electric light is used, showing most 
of the material and apparatus required in ordinary microscopic work. It is 
preferable to have the centrifuge located on another table or shelf and not 
as here shown. Note arrangement of water bottle, waste jar and slide rest 
across it. Note that the tip of the tube from the water bottle is about one 
inch above the slide rest. Have yours so arranged. The tube may be all 
rubber except the bent glass tube that goes to the bottom of the bottle and 
the tip, instead of the intermediate glass tubing as here shown. 



four feet is high enough for the water bottle to be 
placed. The balance of the tube may be rubber, 
except the tip. In ordering, specify "water bottle, 
tubing and pinch cock, described in Clinical Labora- 
tory Diagnosis, Bass and Johns, Fig. 130." 



APPENDIX 



165 



12. 1 diluting pipette for counting blood, 1 to 100, some- 

times called red cell pipette. Fig. 32. 

13. 1 Bass counting chamber, Fig 33. Bausch & Lomb 

Optical Co. and other makers. 
200 c.c. Toison's fluid, p. 35. 



14 
15. 
16. 



18 
19 



Tallquist hemoglobin scale. Fig. 54. 

bottle (2 drams) suspension of typhoid bacilli for 
making Bass-Watkins agglutination test for typhoid. 
See p. 65. Dr. F. M. Johns, P. O. Box 770, New 
Orleans, will furnish this for $1.00 — cash in advance. 
17. 1 medicine dropper, plain (for use in making typhoid 
agglutination test). 

box ordinary hardwood toothpicks, best quality. 

dozen test-tubes, lip less, 12 mm. x 115 mm., outside 
measure. Must be of good quality. If you expect 
to make the modified Wassermann test described in 
this book, you should get 3 dozen of these tubes. 
No other test-tubes are required for the work de- 
scribed in this book. 
20. 1 micro Bunsen burner and 3 feet of pure gum tubing 
suitable for it. See Fig. 131. Don't get the large 
regular size Bunsen burner. If you have not gas 
connections in your laboratory, get an alcohol lamp 
with glass cap to fit over the burner, in place of the 
Bunsen burner. 




Fig. 131. — Micro burner, much better than the larger Bunsen burners. 

21. 1 Urinometer, ordinary form. Fig. 58. 

22. 1 tube litmus paper strips, red. 

23. 1 tube litmus paper strips, blue. 

24. 100 c.c. glacial acetic acid. 



166 APPENDIX 

25. 100 c.c. potassium ferrocyanide solution, 10%. See 

p. 72. 

26. 100 c.c. Fehling's alkaline solution. See p. 74. 

27. 100 c.c. Fehling's copper sulphate solution. See p. 74. 

28. 1 (better 2) graduated pipette, 1 c.c, graduated in 

hundredths. 

29. 50 c.c. hydrochloric acid, C.P. 

30. 50 c.c. chloroform. 

31. 10 grms. sodium nitro-prusside. (This is used only in 

testing urine for acetone.) 

32. 1 hand centrifuge (Fig. 69), with Cornell shields (do 

not accept any other) and rubber washers in these. 
See p. 79. If electricity is available, it is better to 
get an electric centrifuge. Get the Purdy centri- 
fuge, also with Cornell shields. (Fig. 67.) In order- 
ing state whether for direct or indirect current and 
whether for 110 or 220 volt current. 

33. 30 c.c. dimethylaminoazobenzol solution, 0.2% in al- 

cohol. See p. 87. Indicator in testing gastric con- 
tents. 

34. 30 c.c. phenolphthalein solution, 0.2% in alcohol. See 

p. 87. Indicator in testing gastric contents. 

35. 100 c.c. decinormal sodium hydroxide solution. See p. 

88. Should be kept in glass stoppered bottle. Used 
in testing gastric contents. 

36. 1 glass funnel, 2% in. 

37. 10 gm. benzidin (crystals). 

38. 1 Petri dish with cover, 4 in. 

39. 100 c.c. carbol-fuchsin Czaplewsky's formula. Accept 

no other. See p. 115. 

40. 100 c.c. sulphuric acid solution, 2%%. See p. 117. 

41. 6 Bass diphtheria culture tubes, hermetically sealed. 

Accept no other. See p. 127. (H. K. Mulford k 
Co. or Parke, Davis & Co.) 

42. 1 platinum loop in glass rod handle, No. 26 wire. See 

p. 131. 

43. 100 c.c. Loffler's methylene blue solution. See p. 134. 



APPENDIX 



167 



44. 50 c.c. earbol-gentian violet Czaplewsky's formula. Ac- 

cept no other. See p. 136. 

45. 50 c.c. Gram's iodine solution. See p. 136. 

46. 50 c.c. safranin solution, 1%. See p. 138. 

47. 1 bottle Gunther- Wagner Liquid Pearl India Ink. 

You will need this only provided you do not get a 
darkfield condenser, as you should do. 

48. Funnel stop for your oil immersion objective if you 

get a darkfield condenser. Figs. 109 and 111. This 
should be ordered for your particular microscope 
from the manufacturers. Give them the factory num- 
ber of your microscope and the objective. 

49. Antigen for making complement fixation test for syph- 

ilis, described in this book, if you expect to make this 
test. See p. 150. 

50. 1 All glass syringe, 5 c.c. (or 10 c.c), with platino- 

iridium needle, No. 24. 

51. 2 (1 if you don't attempt serum test for syphilis) test- 

tube racks to accommodate 10 or 12 half -inch test- 
tubes (Fig. 127), or bore them in a block of wood. 
Fig. 125 shows a block bored with different size 
holes, and suggests the possibilities. 




Fig. 132. — Proper drop bottle for stains, reagents, etc. 

52. 12 "TK" drop bottles, with flat top. (Fig. 132.) Ac- 
cept no other. (Fig. 133.) 30 c.c. These are for 
your stains and reagents. 



168 



APPENDIX 



53. 30 c.c. peroxide of hydrogen, to use in test for occult 
blood. (You may already have this in your office 
for other purposes.) 




Fig. 133. — Two kinds of improper drop bottles. 

54. 100 c.c. saturated aqueous solution of ammonium sul- 
phate. 



INDEX 

PAGE 

Abscess of liver, leucocyte count in 57 

Acetone in urine, test for 78 

Actinomycosis, leucocyte count in 55 

Addison's disease, leucocyte count in 55 

Agglutination test for para-typhoid 65 

for typhoid 69 

Albumin in urine, test for 71 

Amebae, collection of specimens for examination for/ 102 

differentiation of pathogenic from non-pathogenic 104 

examination of unstained material for 103 

examination for, in dysentery 102 

interpretation of examination for 106 

technic of staining 104 

Anemia, pernicious, leucocyte count in 55 

post-hemorrhagic, leucocyte count in 55 

Anisocytosis 53 

Antigen in complement fixation test for syphilis 149 

dose 149 

source of supply 149 

Apparatus required in testing blood for syphilis 155 

Appendicitis, leucocyte count in 55 

Arthritis, acute, leucocyte count in 55 

Ascaris infection, leucocyte count in 57 

Ascaris lumbricoides, ova of in feces 98 

Asthma, leucocyte count in 55 

Bacteria, staining reaction, motility and morphology of Ill 

in cerebrospinal fluid 124 

morphological classification 109 

Basophiles 26 

Bass counting chamber 34 

Bass diphtheria culture tube 127 

Blood, collection of for test for syphilis 151 

obtaining for all microscopic examinations 12 

occult in feces, test for 106 

occult in gastric contents, test for . 88 

sending specimens by mail to be tested for syphilis 155 

Blood spreads, keeping unstained 22 

labeling 18 

making 17 

Blood staining 22 

"Blood sticker," a good 12 

Bronchiectasis, leucocyte count in 55 

Bronchitis, leucocyte count in 55 

Burns, leucocyte count in 56 

169 



170 INDEX 

PAGE 

Carbol-fuchsin and methylene blue stain for pus and exudates, 

technic 137 

Carbol-fuchsin stain, Czaplewsky's 115 

Carbol- gentian violet stain, Czaplewsky's 138 

Carcinoma, leucocyte count in 56 

Casts, in urine 83 

Centrifuge, use of in examining feces 92 

use of in examining urine . 79 

Cerebro-spinal fluid, cells in 124 

interpretation of examination of „ . 126 

Chancre, scraping to get material for examination 142 

Children, percentage of leucocytes in 31 

Chlorosis, leucocyte count in 56 

Cholangitis, leucocyte count in 56 

Cholecystitis, leucocyte count in 56 

Cholelithiasis, leucocyte count in 56 

Cholera, Asiatic, leucocyte count in 56 

Cirrhosis of liver, leucocyte count in 56 

Color index of blood 51 

interpretation of 51 

Complement fixation test for syphilis, interpretation of 160 

modified method of making 1 19 

Condenser, the Abbe 7 

Counting chamber for blood cells, Bass' 34 

Counting leucocytes 44 

Culture tubes for diphtheria, Bass' 127 

Cylindroids, in urine 84 

Cystitis, acute, leucocyte count in 56 

Czaplewsky's formula for carbol-fuchsin 115 

carbol-gentian violet 141 



Darkfield condenser, use of 143 

Darkground illumination 143 

Dengue, leucocyte count in 56 

Diabetes, leucocyte count in 56 

Digestion, heavy meal, leucocyte count during 56 

Diphtheria, examination of culture for 130 

incubation of culture , . 130 

leucocyte count in 56 

method of making culture 128 

principles of laboratory diagnosis of 127 

Diphtheria bacilli, description of 134 

staining 133 

Diphtheria culture, interpretation of 135 

making preparation from 131 

staining preparation from 133 



Eclampsia, leucocyte count in 56 

Endamebae. (See Amebee.) 



IXDEX 171 

PAGE 

Endocarditis, leucocyte count in 57 

Eosinophils 25 

Epididymitis, gonorrhoea^ leucocyte count in 57 

Erysipelas, leucocyte count in 57 

Erythrocyte counts, calculating the number per cmm. in 49 

interpretation of 51 

total 48 

Erythrocytes, abnormal or pathological 53 

basophilic 54 

method of counting 48 

pathological interpretation of 54 

stippled or granular 54 



Feces, collection of specimens of 90 

concentration of ova in by means of centrifuge . . . 92 

examination of for intestinal parasite ova and larvae 90 

larvae of uncinaria and strongyloides in 100 

making preparation for microscopic examination 91 

method of examining slide preparation of 95 

occult blood in, interpretation of 107 

occult blood in, test for 106 

ova of ascaris in 98 

ova of hymenolepis nana in 99 

ova of oxyuris vermicularis in 99 

ova of tenia in 99 

ova of trichuria in 98 

ova of uncinaria in 97 

Fehling's solution 75 

Filariasis, leucocyte count in 57 

Funnel stop 145 



Gametes 63 

Gametocytes 63 

Gastric contents, examination of 87 

interpretation of examinations of 88 

test for free HC1 in 87 

test for occult blood in 88 

test for total acidity in 87 

Gastritis, leucocyte count in 57 

Gland puncture in diagnosis of syphilis 148 

Gonococci, carbol-fuchsin and methylene blue stain for 137 

description of 140 

Gram's stain for 137 

interpretation of examination for 140 

in urine 135 

Gonorrhoea, in females 136 

laboratory diagnosis of 136 

leucocyte count in 57 

obtaining material and making preparation for examination in. . 136 



172 INDEX 

PAGE 

Gout, leucocyte count in 57 

Gram's solution, formula 138 

Gram's staining method 137 

Guinea-pig, bleeding 150 

Helminthiasis, leucocyte count in 57 

Hemoglobin, estimation of . . . . 50 

per cent., interpretation of 51 

scale — Tallquist's 50 

Hepatitis, leucocyte count in 57 

Hodgkin's disease, leucocyte count in 57 

Hookworm, infection, leucocyte count in 57 

ova of in feces . . • 97 

Hymenolepis nana, ova of in feces 99 

Illumination, darkground 143 

Incubator or waterbath, for complement fixation tests 156 

India ink, use of in examining for spirochetes 146 

Indican in urine, test for 77 

Influenza, leucocyte count in 57 

Intestinal obstruction, leucocyte count in 57 

parasite infections, eosinophiles in 32 

Kala-azar, leucocyte count in 58 

Lamp, Mazda, as source of light in use of microscope 5 

Larvae, differentiation between uncinaria and strongyloides 100 

of strongyloides 100 

of uncinaria 100 

Lead poisoning, stippled erythrocytes in 53 

Lenses, cleaning 11 

oil immersion, use of 10 

Lepra bacilla, description of 121 

interpretation of examination for 121 

staining 121 

Leprosy, microscopic diagnosis of 120 

obtaining material and making preparation from for examina- 
tion 120 

Leucocyte count, differential 26 

influence of infection with pyogenic bacteria upon 31 

interpretation of , 30 

number cells necessary to count in 30 

Leucocyte count, total, acid solution as diluting fluid in making. . 36 

apparatus and material required 34 

calculating number per cmm. in 46 

cleaning pipette 37 

interpretation of 46 

Leucocyte count, making the dilution in . 37 

making preparation for 39 

Toison's solution as diluting fluid in making 35 



INDEX 173 

PAGE 

Leucocytes, abbreviation to be used in making differential count of. 27 

abnormal or pathological 33 

description of found in normal blood 24 

interpretation of presence of pathological 34? 

large mononuclear 24 

percentage of different, in normal adults 30 

percentages in children 31 

polymorphonuclear basophilic 26 

polymorphonuclear eosinophilic 25 

polymorphonuclear neutrophilic 25 

small mononuclear 24 

variations in the proportion of 31 

Leukemia, leucocyte count in 58 

stippled erythrocytes in 54 

Light, regulation of in use of the microscope 8 

source of in use of the microscope 6 

Loffler's methylene blue stain, formula 137 

Lumbar puncture 122 

Malaria, leucocyte count in 58 

Malaria, plasmodia, description of 61 

differentiation of 63 

examination for 61 

interpretation of examination of blood for 64 

making preparations to be examined for 61 

staining 62 

time to examine for 61 

Mastoiditis, leucocyte count in 59 

Mazda lamp, gas-fitted 144 

Measles, leucocyte count in 58 

Megaloblasts 52 

Megalocytes 52 

Meningitis, cerebro-spinal 125 

leucocyte count in 58 

obtaining material for examination for 125 

Meningococci, in cerebro-spinal fluid 124 

Merozoites 62 

Methylene blue stain, Loffler's, making 137 

Microscope, adjustment of mirror in use of 7 

care of stand of 11 

focussing 10 

light in the use of 6 

model of Bausch & Lomb make recommended 1 

model of Leitz make recommended 1 

model of Spencer make recommended 1 

selection of a 1 

use and care of the 1 

Mumps, leucocyte count in 58 

Myelocytes, basophilic 33 

eosinophilic 33 

neutrophilic 33 



174 INDEX 

PAGE 

Myxedema, leucocyte count in 58 

Nephritis, leucocyte count in 58 

Neutrophiles 25 

Newton's color rings, looking for in blood preparation 42 

Normoblasts 53 

Occult blood, test for in feces 107 

test for in gastric contends 87 

Orchitis, gonorrhoea!, leucocyte count in 57 

Otitis media, leucocyte count in 59 

Oxyuris infection, leucocyte count in . 57 

vermicularis, ova of in feces 99 

Pellagra 59 

Pneumonia 59 

Poikilo^ytosis 53 

Polychromatophilia 53 

Pregnancy 59 

Pyelitis 59 

Pyelonephritis 59 

Red blood cells. (See Erythrocytes.) 

Round worm, ova of in feces 99 

Safranin as a counterstain " 140 

Sarcomatosis, leucocyte count in 59 

Scarlatina, leucocyte count in . . 59 

Schizogony 62 

Schizonts 62 

Scurvy, leucocyte count in 59 

Septicemia, leucocyte count in 59 

Simon's septic factor 33 

Slides, cleaning 18 

Smallpox, leucocyte count in 59 

Spinal fluid . 122 

cell counts in 125 

interpretation of findings in . 125 

meningococci in 124 

obtaining material for examination of 122 

other bacteria in 124 

pneumococci in 124 

test of for globulin increase 125 

tubercle bacilli in . 124 

Spirocheta. (See Treponema.) 

dentium 146 

refringens 147 

Spirochetes, movements of . 145 

Sputum, collecting specimens of • • 112 

examination of for tubercle bacilli 112 

interpretation of examination of 121 

Stain, Wright's 22 



INDEX 175 

PAGE 

Strongyloides infection, leucocyte count in 57 

Sugar in urine, test for 74 

Syphilis 141 

apparatus required in testing blood for 155 

collection of blood to be tested for 141 

examination for Treponema pallida in 148 

gland puncture in diagnosis of 148 

interpretation of complement fixation test for 160 

leucocyte count in 59 

sending specimens of blood by mail to be tested for 155 

Wassermann serum test in 149 

Tallquist's hemoglobin scale 49 

Tenia infection, leucocyte count in 57 

ova of in feces 99 

Test meal, in gastric analysis 87 

Test-tube block, wood 156 

Toison's fluid 35 

Tonsillitis, leucocyte count in 59 

Total acidity, in gastric contents 87 

Treponema macrodentium 147 

Treponema microdentium 147 

Treponema pallida, differentiation of from other spirochetes 148 

examination for 141 

interpretation of examination for 160 

Treponema pertenuis 148 

Trichinosis, leucocyte count in 60 

Trichuria trichuris, ova of in feces 98 

Tricocephalus. (See Trichuria trichuris.) 

infection, leucocyte count in 57 

Tubercle bacilli, description of 124 

in cerebrospinal fluid 125 

in sputum 114 

making preparation for examination for 115 

staining 116 

Tuberculosis, leucocyte count in 60 

Typhoid, agglutination test for 65 

agglutination test, interpretation of 67 

leucocyte count in _. 60 

Typhus fever, leucocyte count in 60 

Uncinaria, ova of in feces 97 

Uremia, leucocyte count in 60 

Urine, albumin in, test for, qualitative 71 

albumin in, test for, quantitative 72 

acetone in, test for 78 

casts in 83 

collection of specimens of for examination 70 

cylindroids in 84 

epithelial cells in 83 

indican in, test for 77 

interpretation of examinations of 85 



176 INDEX 

PAGE 

Urine, microscopic examination of , 73 

preservation of specimens of 70 

pus cells in 83 

quantitative 76 

reaction of 71 

red blood cells in 82 

specific gravity of 71 

sugar in, test for, qualitative 74 

Wassermann reaction, interpretation of 160 

Whipworm, ova of in feces 99 

Whooping cough, leucocyte count in 60 

Women, small mononuclear leucocytes in blood of 31 

Wright's stain * 22 

Yellow fever, leucocyte count in 60 



